Screening methods for transfusion related acute lung injury

ABSTRACT

The invention relates to the discovery that HNA-3a and HNA-3b are antigens within a polypeptide sequence that is highly similar to the CTL2 amino acid sequence. This invention provides methods and kits for screening for HNA-3a and HNA-3b specific antibodies, HNA-3a and HNA-3b polypeptides and HNA-3a and HNA-3b nucleic acids in a sample of a biological tissue intended for transplantation.

This application is a divisional of U.S. patent application Ser. No.12/553,487, filed Sep. 3, 2009 now U.S. Pat. No. 8,084,216, which claimspriority to German Application No. 10 2008 045 696.9 filed Sep. 4, 2008,which are herein incorporated by reference in its their entirety.

FIELD OF INVENTION

The invention relates to the identification of the polypeptide sequencesof HNA-3a and HNA-3b antigens which are implicated in the occurrence oftransfusion related acute lung injury syndrome (TRALI). This inventionprovides methods and kits for screening for HNA-3a and HNA-3b specificantibodies, HNA-3a and HNA-3b polypeptides, and HNA-3a and HNA-3bnucleic acids in a sample of a biological tissue intended fortransplantation or transfusion. The invention also relates to methodsand kits for determining whether donor tissues intended for transplantor transfusion will induce TRALI. The invention further provides formethods and kits for determining the susceptibility of a humantransplant or transfusion recipient of developing TRALI

BACKGROUND

Antibodies to human neutrophil-specific antigens (HNA) were shown tocause clinical complications such as pulmonary transfusion reactions andin some cases transfusion related acute lung injury (TRALI) (Popovsky etal. Am. Rev. Resp. Dis. 128(1): 185-9, 1983) or causing neonatalalloimmune neutropenia (NAIN) (Bux, et al. Transfus. Med. 2(2): 143-9,1992). Therefore, detection of HNA specific antibodies has importantclinical applications.

TRALI is a life-threatening transfusion complication and is one of themost frequent causes of transfusion-related death in the United States.TRALI is the second most frequent transfusion-related cause of death inEurope after administration of ABO-incompatible stored blood (Holness etal. Transfus Med. Rev. 18: 184-188, 2004). The risk of developing TRALIas a complication of blood transfusion is at least 2000-times higherthan contracting an HIV- or hepatitis C infection.

TRALI is defined as a clinical entity consisting of sudden acuteshortness of breath within six hours after blood transfusion, connectedwith bilateral lung infiltrations (lung edema) with no indication ofcardiac insufficiency or volume overload (European HaemovigilanceNetwork (EHN). Definitions of Adverse Transfusion Events available onthe EHN web site.

TRALI syndrome is difficult to diagnose, because initially it often doesnot differ from a transfusion-independent lung insufficiency (ALI) orits maximum variant ARDS (“acquired respiratory distress syndrome”)(Popovsky & Moore, Transfusion 25: 573-577, 1985). Symptoms of TRALIinclude hypoxemia, tachycardia, hypotension, cyanosis and fever. Often,TRALI is not recognized or misdiagnosed in the clinic because thesymptoms are often attributed to other causes, such as fluid overload.TRALI has been associated with the transfusions of all plasma containingblood components, including whole blood, red blood concentrates, freshfrozen plasma, whole blood derived from platelets, pooled platelets,intravenous gamma-globulin, cryoprecipitate, stem cells andgranulocytes. TRALI is an injury to the pulmonary microvascular; andtherefore, treatment focuses on respiratory support and saline infusion.

TRALI is an immune-related disorder that is primarily associated withantibodies specific for HNA, granulocyte- and human leukocyte antigens(HLA) Class I. Other factors that have induced TRALI in transfusionrecipients include biologically active lipids and HLA Class IIantibodies. In most cases, antibodies of the donor (in the donor plasma)are transferred with the stored blood and then react with the leucocytes(granulocytes) of the recipient. The binding of the antibodies to thegranulocytes leads to their activation and partially to aggregation.Through the subsequent release of the microbicidal arsenal from thegranulocytes, the capillary endothelium is damaged which results in lungedema. The immune reaction induces complement-activated granulocytes torelease oxygen radicals and proteases that damage the endotheliumresulting in the extravasation of protein-rich fluid into the pulmonaryalveoli and interstitium. In addition, antibodies within stored bloodwill bind to and activate granulocytes of the recipient resulting in theexpression of adhesion molecules (Uchiyama et al. Transfus. Med. Rev. 8:84-95, 1994), transmigration of granulocytes into the interstitial spacebetween alveolar and vessel endothelium of the lung, and the release ofcytokines, proteases and oxygen radicals (Snyder, Immol Invest. 24:333-9, 1994). These cellular effects cause damage to the capillary wallswith subsequent hyperpermeability. A lung edema develops and 10% of theaffected patients die from this complication.

In TRALI, recipient antibodies rarely react with the granulocytes of thedonor (Bux et al., Br. J. Heamatol. 93: 707-713, 1996). However, therehave also been cases of TRALI that were induced by antibodies in thetransfusion recipient. In very rare cases, anti-IgA-antibodies can alsoinduce TRALI (Saigo et al., J. Int. Med. Res. 27: 96-100, 1999).

Blood donations of multiparous women carry particular risk, because anantibody formation against granulocyte- or HLA-antigen of the child canoccur during the pregnancies Likewise, a patient may be immunizing dueto an earlier transfusion (Voss et al., Anaesthesist 50: 930-932, 2001).Donor plasma that will trigger TRALI cannot be detected clinically.Currently produced erythrocyte concentrates contain very little plasmaand only a few granulocytes, therefore TRALI is most likely to occurafter administration of fresh plasma and platelet concentrates.

In addition to HLA antibodies, antibodies against three differentantigen systems on granulocytes are thought to be responsible forinducing TRALI (Leger et al. Anesthesiology 91: 1529-1532, 1999; Davorenet al, Transfusion 43: 641-645, 2003; Kopko et al. JAMA 287: 1968-1971,2000; Reil et al. Vox Sanguinis (printing, already accessible online),2008. Two of the antigen systems (HNA-1 and HNA-2) are known with regardto their structure and localization. The antigen HNA-2 was characterizedby Prof. Dr. Bux and applied for as a patent (DE 100 28 725 A1). Thethird antigen system, HNA-3 (consisting of the antithetic antigensHNA-3a and HNA-3b), has not been characterized. The antigen HNA-3aoccurs in approximately 95% of the population (Davoren et al,Transfusion 43: 641-645, 2003) and is involved particularly frequentlyin severe courses of TRALI (Reil et al., Vox Sanguinis (printing,already accessible online), 2008).

According to the current report by SHOT (Serious Hazards ofTransfusion), the British notification and evaluation centre forside-effects in blood transfusions, TRALI is the most frequent cause ofa serious side-effect due to transfusion. The report shows a mortalityof 9% for the period 1996-2003 (SHOT), Additional cumulative data1996-2003 is available at the Serious Hazards of Transfusion (SHOT) website). Since 2001, United States Food and Drug Administration likewisereported TRALI as the main cause of transfusion-associated complications(Goldman et al., Transfus. Med. Rev. 19: 2-31, 2005; Boshkov, Vox Sang.83: 299-303, 2002).

Currently, most blood and tissue donors have not been HNA typed. Thespecialized nature of neutrophil immunobiology, the scarcity of HNAtyping sera and the need to test fresh neutrophils places restraints ontyping HNA compatible blood components. A high percentage of cases ofTRALI are caused by blood donated by females, particularly multiparousfemales, and from the transfusion of fresh frozen plasma. Proposedcurrent solutions for reducing the incidence of TRALI include theexclusion of all females as donors, to exclude multiparous (three ormore pregnancies) females as donors, and reducing the transfusion offresh, frozen plasma

Currently, the detection of granulocyte-specific antibodies islaborious; and detection of HLA antibodies in the serum of the blooddonor is not sufficient. The most reliable determination of a TRALI riskcurrently consists in a cross-matching between donor serum and patientleucocytes. This test can only be carried out in specializedlaboratories (Voss, Anaesthesist 50: 930-932, 2001) which are notsuitable for donor screening. Other strategies are currently directed toa more restrictive donor management (Mair et al., Crit. Care Med. 34:137-143, 2006) (as described above). This is not acceptable because theexclusion of women from blood donation after a pregnancy leads to aserious reduction in the amount of stored blood.

The exclusion of female donors was investigated systematically inCanada. Through the exclusion of multiparous female donors, 12% of allblood donations would be omitted from the Canadian Blood Service(Goldman et al Transfus. Med. Rev. 19: 2-31, 2005). According to somestudies, implementing such a strategy would exclude every thirdpotential female donor. (Densmore et al., Transfusion 39: 103-106,1999). An alternative strategy would be the testing of all stored bloodfor granulocyte-specific antibodies. Currently, this technically cannotbe carried out. Other strategies for processing the blood components areproposed, but these strategies would involve new risks such as bacterialcontaminations and due to their time requirement are only suitable forplanned transfusions, and not for those in emergencies (Mair et al.,Crit. Care Med. 34: 137-143, 2006). Furthermore, evidence is lacking asto whether such strategies can actually reduce the incidence of TRALI.

Human neutrophil antigens are also known as neutrophil-specific antigensor HNA. Currently there are 5 HNA antigen systems: HNA-1, HNA-2, HNA-3,HNA-4 and HNA-5. Alleles for HNA-1, 2, 4 and 5 were identified and thecorresponding glycoproteins were characterized; however, the allele forHNA-3 remains unknown (reviewed by Stroncek, ASHI Quarterly 2004). Thereare three HNA-1 antigens (HNA-1a, HNA-1b and HNA-1c) that are expressedsolely on neutrophils and are located on low affinity Fc-γ receptorIIIb. The HNA-2 system has one well established antigen (HNA-2a). HNA-2is only expressed on neutrophils and neutrophil precursors and islocated on the glycoprotein CD177 (NB1 gp). HNA-4 and HNA-5 are locatedon the β2 integrin. HNA-4 is expressed on granulocytes, monocytes andlymphocytes. (See Stroncek, ASHI Quarterly 2004)

The HNA-3 system has one known antigen, HNA-3a, which is also known as5b. HNA-3 is expressed on neutrophils, lymphocytes, platelets,endothelial cells, kidney, spleen and placenta cells, and is known to belocated on a 70 to 95 kDa neutrophil glycoprotein. (See Stroncek, ASHIQuarterly 2004). The gene for HNA-3a has not been cloned and the natureand function of glycoprotein was previously unknown. Therefore, currentdetection of HNA-3 antibody is only based on non-specific assays, suchas agglutination tests (Lalezari & Bernard, Transfusion 5: 135-42, 1965)or GIFT-FC assay (Davoren et al., Transfusion 43(5): 641-5, 2003).

The presumed allele of HNA-3, also known as 5b, has a gene frequencyabout 0.82 (Van Leeuwen et al. Vox Sang 9: 431-46, 1964). It was alsoreported to have a 0.66 gene frequency (Lalezari & Bernard, Transfusion5: 135-42, 1965). The protein of 5b was reported to have a molecularweight of 70 to 95 kD (De Haas et al, Transfusion 40(2): 222-7, 2000),yet the 5b gene has not been cloned and the nature and function of theprotein remain unknown.

Of interest to the present invention is CTL2 is a 706 amino acidmembrane-spanning protein (about 80.152 kD) that comprises 10 helicaltransmembrane domains. This protein is also known as which is also knownas SLC44A2, DKFZp666A071 2, FLJ44586 2 and PP1292 and is known to beinvolved in choline transport within the inner ear and is expressed oninner ear supporting cells. The gene encoding CTL2 is located onchromosome 19p13. In addition, the antigen, Inner Ear Supporting CellAntigen (IESCA) is known to be a CTL2 protein which is reactive with anautoantibody associated with autoimmune sensorineural hearing loss(AISNHL).

Currently, the methods of screening and typing transplant tissue ortransfusions for HNA antibodies that induce TRALI are inadequate andproblematic. In addition, excluding a large portion of the humanpopulation from donating blood and tissue is an extreme solution.Therefore, a strong need exists for the development of methods ofscreening for HNA antigens.

It was therefore an object of the present invention to clarify theprotein- or DNA sequences of the human neutrophil antigen-3a or -3b(HNA-3a, HNA-3b) involved in TRALI and to provide the correspondingsequences.

SUMMARY OF INVENTION

The present invention relates to the discovery that HNA-3a is within theamino acids sequence of SEQ ID NO: 1 and HNA-3b is within the amino acidsequence of SEQ ID NO: 3b, and these amino acid sequence are highlysimilar to that of choline transporter-like protein 2 (CTL2).

A single nucleotide polymorphism (SNP) within codon 154 on anextracellular loop is crucial (SNP rs2288904), as this SNP is thedifference between HNA-3a and HNA-3b. The polynucleotide encoding HNA-3ahas a “G” (guanine) at position 461 and as a result encodes an “R”(Arginine, Arg) at position 154 of the HNA-3a amino acid sequence, andtherefore represents the HNA-3a allele. The polynucleotide encodingHNA-3b has an “A” (adenine) at position 461 and as a result encodes a“Q” (glutamine, Gln) at position 154 of the amino acid sequence, andtherefore represents the HNA-3b allele.

It is proposed that the existence of the SNP within codon 154 of theHNA-3 gene results in a portion of the population which may generatealloantibodies to HNA-3a or HNA-3b if exposed to the opposing HNA-3antigen. This difference allows for portions of the population to haveone of two anti-HNA-3 specific antibodies and when exposed to blood ortissue containing a foreign HNA-3 antigen will induce transfusionrelated acute lung injury (TRALI) in the recipient.

The invention provides for methods of detecting an HNA-3a specificantibody in a biological sample comprising a) obtaining a biologicalsample, b) contacting the biological sample with a cell transformed ortransfected to express the HNA-3a polypeptide of SEQ ID NO: 1 or afragment thereof to form a complex with HNA-3a in the sample, and c)detecting the complex, wherein the presence of the complex indicatesthat the biological sample contains HNA-3a specific antibodies.

The preceding methods may be carried out with an antigenic fragmentcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

The invention also provides for methods of detecting an HNA-3b specificantibody in a biological sample comprising a) obtaining a biologicalsample, b) contacting the biological sample with a cell transformed ortransfected to express the HNA-3b polypeptide of SEQ ID NO: 2 or afragment thereof to form a complex with HNA-3b in the sample, anddetecting the complex, wherein the presence of the complex indicatesthat the biological sample contains HNA-3b specific antibodies.

The preceding methods may be carried out with an antigenic fragmentcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24 and SEQ ID NO: 26.

The invention contemplates using any cell type including those which donot endogenously express HNA-3a or HNA-3b such as B-cells, CHO cells orinsect cells, so that the cell expresses a heterologous HNA-3a or HNA-3polypeptide. The term “heterologous” refers to cytologic elements, e.g.DNA or protein that are derived from a different species or differentcell type.

The invention also contemplates using a cell that expresses low levelsof HNA-3a or HNA-3b and increasing expression of the endogenous proteinby inserting heterologous promoters or enhancers, or increasing copynumber of the HNA-3a or HNA-3b gene. Exemplary cells that may be usedinclude EB-3 cells and K-562 cells.

The invention also provides for methods of detecting an HNA-3a specificantibody in a biological sample comprising a) obtaining a biologicalsample, b) contacting the biological sample with an aptamer that mimicsan antigenic fragment of the HNA-3a polypeptide of SEQ ID NO: 1 to forma complex with the HNA-3a specific antibodies in the sample, and c)detecting the complex, wherein the presence of the complex indicatesthat the biological sample contains HNA-3a specific antibodies. Thesemethods may be carried out with aptamers that mimic an antigenicfragment comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

The invention also provides for methods of detecting an HNA-3a specificantibody in a biological sample comprising a) obtaining a biologicalsample, b) contacting the biological sample with an aptamer that mimicsan antigenic fragment of the HNA-3b polypeptide of SEQ ID NO: 2 to forma complex with the HNA-3b specific antibodies in the sample, and c)detecting the complex, wherein the presence of the complex indicatesthat the biological sample contains HNA-3b specific antibodies. Thesemethods may be carried out with aptamers that mimic an antigenicfragment comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 26.

The invention provides for methods of screening for HNA-3a and/or HNA-3bspecific antibodies in donor tissue intended for transplants ortransfusion in order to determine whether the donor tissue, as a resultof the presence of the HNA-3a or HNA-3b specific antibodies, will induceTRALI or graph versus host disease (GVHD) in a human recipient thatexpresses the HNA-3a or HNA-3b.

In one embodiment, the invention provides for methods of determiningwhether a donor tissue intended for transplant or transfusion willinduce TRALI or GVHD in a human recipient wherein the human recipientexpresses the HNA-3a antigen comprising a) obtaining a sample of thetissue intended for transplant or transfusion in the human subject, b)contacting the sample with a polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or an antigenic fragment thereof to form acomplex with HNA-3a specific antibodies in the sample, and c) detectingthe complex, wherein the presence of the complex indicates that thedonor tissue is likely to induce TRALI or GVDH in a human recipient thatexpresses the HNA-3a antigen. These methods may be carried out the withan antigenic fragment of the HNA-3a amino acid sequence (SEQ ID NO: 1)selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQID NO: 42.

In another embodiment, the invention provides for methods of determiningwhether a donor tissue intended for transplant or transfusion willinduce TRALI or GVHD in a human recipient wherein the human recipientexpresses the HNA-3b antigen comprising a) obtaining a sample of thetissue intended for transplant or transfusion in the human subject, b)contacting the sample with a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or an antigenic fragment thereof to form acomplex with HNA-3b specific antibodies in the sample, and c) detectingthe complex, wherein the presence of the complex indicates that thedonor tissue is likely to induce TRALI or GVHD in a human recipient thatexpresses the HNA-3b antigen. These methods may be carried out withantigenic fragments of the HNA-3b amino acid sequence (SEQ ID NO: 2)selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO:26.

In a further embodiment, the methods of determining whether a donortissue intended for transplant or transfusion will induce TRALI or GVHDin a human recipient wherein the human recipient expresses the HNA-3a orHNA-3b antigen, wherein in addition to detecting HNA-3a or HNA-3bspecific antibodies, the methods further comprise one or more of thefollowing steps: contacting the sample with Fc-γ receptor IIIbpolypeptide or an antigenic fragment thereof to form a complex withHNA-1 specific antibodies in the sample, contacting the sample withCD177 polypeptide or an antigenic fragment thereof to form a complexwith HNA-2 specific antibodies in the sample, contacting the sample withCD11b polypeptide or an antigenic fragment thereof to form a complexwith HNA-4 specific antibodies in the sample, contacting the sample withCD11a polypeptide or an antigenic fragment thereof to form a complexwith HNA-5 specific antibodies in the sample, or contacting the samplewith an HLA antigen to form a complex with HLA specific antibodies inthe sample, and detecting the complex, wherein the presence of any ofthe complexes indicates that the sample is likely to induce TRALI orGVHD in a human recipient.

The invention also provides for methods of screening a transplant ortransfusion recipient for HNA-3a and/or HNA-3b specific antibodies. Thisscreening is of interest because if a donor tissue intended fortransplants or transfusion comprises the HNA-3a or HNA-antigen, it islikely that the transplanted or transfused tissue will be rejected ifthe recipient comprises antibodies to the corresponding antigen. Inrejection, the recipients' antibodies will bind to and target the tissueas foreign which will result it destruction by its immune system.

In one embodiment, the invention provides for methods of determining thesusceptibility of a human transplant or transfusion recipient forrejecting transplanted or transfused tissue, wherein the donor tissuecontains HNA-3a polypeptide or an antigenic fragment thereof, comprisinga) obtaining a biological sample from the human transplant ortransfusion recipient prior to transplantation or transfusion, b)contacting the biological sample with polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or an antigenic fragment thereof to form acomplex with HNA-3a specific antibodies in the biological sample, and c)detecting the complex, wherein the presence of the complex in thebiological sample indicates that the human transplant or transfusionrecipient is susceptible for rejecting the transplanted or transfusedtissue. These methods may be carried out the with an antigenic fragmentsof the HNA-3a amino acid sequence (SEQ ID NO: 1) selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

In another embodiment, the invention provides for methods of determiningthe susceptibility of a human transplant or transfusion recipient forrejecting transplanted or transfused tissue, wherein the donor tissuecontains HNA-3b polypeptide or an antigenic fragment thereof, comprisinga) obtaining a biological sample from the human transplant ortransfusion recipient prior to transplantation or transfusion, b)contacting the biological sample with polypeptide comprising the aminoacid sequence of SEQ ID NO: 2 or an antigenic fragment thereof to form acomplex with HNA-3b specific antibodies in the biological sample, and c)detecting the complex, wherein the presence of the complex in thebiological sample indicates that the human transplant or transfusionrecipient is susceptible for rejecting the transplanted or transfusedtissue. These methods may be carried out with an antigenic fragments ofthe HNA-3b amino acid sequence (SEQ ID NO: 2) selected from the groupconsisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 26.

In a further embodiment, the invention provides for methods ofdetermining the susceptibility of rejecting transplanted or transfusedtissue, wherein the donor tissue contains HNA-3a or HNA-3b polypeptideor an antigenic fragment thereof, wherein in addition to detecting HNA3aor HNA-3b specific antibodies, the methods further comprise one of moreof the following steps: contacting the biological sample with Fc-γreceptor IIIb polypeptide or an antigenic fragment thereof to form acomplex with HNA-1 specific antibodies in the biological sample,contacting the biological sample with CD177 polypeptide or an antigenicfragment thereof to form a complex with HNA-2 specific antibodies in thebiological sample, contacting the biological sample with CD11bpolypeptide or an antigenic fragment thereof to form a complex withHNA-4 specific antibodies in the biological sample, contacting thebiological sample with CD11a polypeptide or an antigenic fragmentthereof to form a complex with HNA-5 specific antibodies in thebiological sample, or contacting the biological sample with an HLAantigen to form a complex with HLA specific antibodies in the biologicalsample, and detecting the complex, wherein the presence of any of thecomplexes in the biological sample indicates that that the humantransplant or transfusion recipient is susceptible for rejecting thetransplanted or transfused tissue, wherein the donor tissue contains anyof HNA-1, HNA-2, HNA-3a, HNA-3b, HNA-4, HNA-5, and HLA.

Any of the preceding methods may be carried out with aptamers whichmimic the HNA-3a or HNA-3b epitope and therefore bind to HNA-3a orHNA-3b specific antibodies.

The invention provides for methods of screening for HNA-3a and/or HNA-3bantigen in donor tissue. Screening for HNA-3a or HNA-3b antigen isimportant when a transplant or transfusion recipient is known to expressHNA-3a or HNA-3b antibodies. The recipient's antibodies may bind to thecells within the donor tissue causing or increasing the risk ofrejection of the transplanted or transfused tissue.

In one embodiment, the invention provides for methods of determiningwhether a donor tissue intended for transplant or transfusion is likelyto be rejected in a human recipient wherein the human recipient hasHNA-3a specific antibodies, comprising a) obtaining a sample of thetissue intended for transplant or transfusion in the human recipient, b)contacting the sample with an antibody that specifically binds to aHNA-3a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 ora fragment thereof to form a complex with HNA-3a in the sample, and c)detecting the complex, wherein the presence of the complex indicatesthat the donor tissue is likely to be rejected in a human recipient thatexpresses HNA-3a specific antibodies.

In another embodiment, the invention provides for methods of determiningwhether a donor tissue intended for transplant or transfusion is likelyto be rejected in a human recipient wherein the human recipient hasHNA-3b specific antibodies, comprising a) obtaining a sample of thetissue intended for transplant or transfusion in the human recipient,contacting the sample with an antibody that specifically binds to HNA-3bpolypeptide comprising the amino acid sequence of SEQ ID NO: 2 or afragment thereof to form a complex with HNA-3b in the sample, anddetecting the complex, wherein the presence of the complex indicatesthat the donor tissue is likely to be rejected in a human recipient thatexpresses HNA-3b specific antibodies.

In a further embodiment, the invention provides for methods ofdetermining whether a donor tissue intended for transplant ortransfusion will induce TRALI or GVHD in a human recipient wherein thehuman recipient has HNA-3a specific antibodies or HNA-3b specificantibodies, wherein in addition to detecting HNA-3a and/or HNA-3bantigen, the method further comprise one or more of the following steps:contacting the sample with an antibody that specifically binds to HNA-1to form a complex with HNA-1 in the sample, contacting the sample withan antibody that specifically binds to HNA-2 to form a complex withHNA-2 in the sample, contacting the sample with an antibody thatspecifically binds to HNA-4 to form a complex with HNA-4 in the sample,contacting the sample with an antibody that specifically binds to HNA-5to form a complex with HNA-5 in the sample, or contacting the samplewith an antibody that specifically binds to HLA antigen to form acomplex with an HLA in the sample, and detecting the complex, whereinthe presence of any of the complexes indicates that the sample is likelyto induce TRALI or GVHD in a human recipient.

In any of the foregoing methods, the antibodies may comprise a labelselected from the group consisting of a radioactive label, fluorescentlabel, enzymatic label, avidin label or biotin label. In addition, inany of the above described methods, the antigen-antibody complex may bedetected with a secondary antibody. The secondary antibodies maycomprise a label selected from the group consisting of a radioactivelabel, fluorescent label, enzymatic label, avidin label or biotin label.

The invention further provides for methods of genotyping the HNA-3a orHNA-3b allele of a transplant or transfusion donor. The term genotypingrefers to detecting the presence of a particular allele, e.g. HNA-3a orHNA-3b, of a human subject. These methods are of interest when theintended transplant or transfusion recipient is known to express HNA-3aor HNA-3b antibodies, and therefore the presence of the HNA-3a or HNA-3bantigen respectively in a transfused or transfected tissue is likely tobe rejected by the recipient. The methods of genotyping may employ theoligonucleotide probes that detect the HNA-3a or HNA-3b allele, or PCRto amplify a nucleotide fragment that comprises the HNA-3a or HNA-3ballele or using sequencing methods standard in the art to detect theHNA-3a or HNA-3b allele.

In one embodiment, the invention provides for methods of determiningwhether a donor tissue intended for transplant or transfusion is likelyto be rejected in a human recipient wherein the human recipientexpresses HNA-3a specific antibodies, comprising a) obtaining a sampleof the tissue intended for transplant or transfusion, b) extractingnucleic acids from the sample, c) contacting the nucleic acids with anoligonucleotide probe that hybridizes to a fragment of SEQ ID NO: 3, andd) detecting hybridization of the probe to the nucleic acids, whereinhybridization of the probe indicates the presence of an HNA-3a nucleicacid and the presence of HNA-3a nucleic acid in the sample indicatesthat the sample is likely to be rejected in a human recipient thatexpresses HNA-3a specific antibodies. These methods may be carried outwith an oligonucleotide probe that comprises a nucleotide sequence thatencodes an amino acid sequence selected from the group consisting of SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ IDNO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

The invention also provide for methods of determining whether a donortissue intended for transplant or transfusion is likely to be rejectedin a human recipient wherein the human recipient expresses HNA-3bspecific antibodies, comprising a) obtaining a sample of the tissueintended for transplant or transfusion, b) extracting nucleic acids fromthe sample, c) contacting the nucleic acids with an oligonucleotideprobe that hybridizes to a fragment of SEQ ID NO: 4, and d) detectinghybridization of the probe to the nucleic acids, wherein hybridizationof the probe indicates the presence of an HNA-3b nucleic acid and thepresence of HNA-3b nucleic acid in the sample indicates that the sampleis likely to be rejected in a human recipient that expresses HNA-3bspecific antibodies. These methods may be carried out with anoligonucleotide probe that comprises a nucleotide sequence that encodesan amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24 and SEQ ID NO: 26.

In a further embodiment, the methods of determining whether a donortissue intended for transplant or transfusion is likely to be rejectedin a human recipient wherein the human recipient expresses HNA-3a orHNA-3b specific antibodies, wherein in addition to detecting thepresence of HNA-3a allele or HNA-3b allele, the methods may comprise oneor more of the following steps: contacting the sample with anoligonucleotide probe that hybridizes to a fragment of SEQ ID NO: 5,contacting the sample with an oligonucleotide probe that hybridizes to afragment of SEQ ID NO: 7, contacting the sample with an oligonucleotideprobe that hybridizes to a fragment of SEQ ID NO: 9, contacting thesample with an oligonucleotide probe that hybridizes to a fragment ofSEQ ID NO: 11, or contacting the sample with an oligonucleotide probethat hybridizes to a fragment of a nucleotide sequence encoding an HLAantigen, and detecting the hybridization of the probe to the nucleicacids, wherein hybridization of any of the probes indicates the presenceof any one of HNA-1, HNA-2, HNA-3a, HNA-3b, HNA-4, HNA-5 or HLA nucleicacid in the sample and the presence of any one of HNA-1, HNA-2, HNA-3a,HNA-3b, HNA-4, HNA-5 or HLA nucleic acid in the sample indicates thatthe sample is likely to be rejected in a human recipient. For example,the invention contemplates that the fragment of HNA-1 (SEQ ID NO: 5)comprises at least one of nucleotide 141, nucleotide 147, nucleotide226, nucleotide 227, nucleotide 277 or nucleotide 349 of SEQ ID NO: 5.

In another embodiment, the invention provides for methods of determiningthe susceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD, wherein the donor tissue contains HNA-3aspecific antibodies, comprising a) obtaining a biological sample fromthe human transplant or transfusion recipient prior to transplantationor transfusion, b) extracting nucleic acids from the biological sample,c) contacting the nucleic acids with an oligonucleotide probe thathybridizes to a fragment of the nucleotide sequence of SEQ ID NO: 1, andd) detecting hybridization of the probe to the nucleic acid, whereinhybridization of the probe to the nucleic acids indicates the presenceof HNA-3a nucleic acid in the biological sample and the presence ofHNA-3a in the biological sample indicates that a human transfusion ortransplant recipient is susceptible for developing TRALI or GVHD. Thesemethods may be carried out with an oligonucleotide probe that comprisesa nucleotide sequence that encodes an amino acid sequence selected fromthe group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

The invention also provides for methods of determining thesusceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD, wherein the donor tissue contains HNA-3bspecific antibodies, comprising a) obtaining a biological sample fromthe human transplant or transfusion recipient prior to transplantationor transfusion, b) extracting nucleic acids from the biological sample,c) contacting the nucleic acids with a oligonucleotide probe thathybridizes to a fragment of the nucleotide sequence of SEQ ID NO: 2, andd) detecting hybridization of the probe to the nucleic acid, whereinhybridization of the probe to the nucleic acids indicates the presenceof HNA-3b nucleic acid in the biological sample and the presence ofHNA-3b in the biological sample indicates that a human transfusion ortransplant recipient is susceptible for developing TRALI or GVHD. Thesemethods may be carried out with an oligonucleotide probe that comprisesa nucleotide sequence that encodes an amino acid sequence selected fromthe group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 26.

The invention further provides for methods of determining thesusceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD, wherein the donor tissue contains HNA-3a orHNA-3b specific antibodies, wherein in addition to detecting thepresence of HNA-3a or HNA-3b allele respectively, the methods mayfurther comprise one or more of the following steps: contacting thesample with an oligonucleotide probe that hybridizes to a fragment ofSEQ ID NO: 5, contacting the sample with an oligonucleotide probe thathybridizes to a fragment of SEQ ID NO: 7, contacting the sample with anoligonucleotide probe that hybridizes to a fragment of SEQ ID NO: 9,contacting the sample with an oligonucleotide probe that hybridizes to afragment of SEQ ID NO: 11, or contacting the sample with anoligonucleotide probe that hybridizes to a fragment of a nucleotidesequence encoding an HLA antigen, and detecting the hybridization of theprobe to the nucleic acids, wherein hybridization of any of the probesindicates the presence of any one of HNA-1, HNA-2, HNA-3a, HNA-3b,HNA-4, HNA-5 or HLA nucleic acid in the sample and the presence of anyone of HNA-1, HNA-2, HNA-3a, HNA-3b, HNA-4, HNA-5 or HLA nucleic acid inthe sample indicates that the sample is likely to induce TRALI or GVHDin a human recipient. For example, the invention contemplates that thefragment of HNA-1 (SEQ ID NO: 5) comprises at least one of nucleotides141, nucleotide 147, nucleotide 226, nucleotide 227, nucleotide 277 ornucleotide 349 of SEQ ID NO: 5.

In any of the preceding methods, the oligonucleotide probes may beaffixed to a substrate selected from the group consisting of membranes,filters, beads and chips. In addition, the invention provides formethods wherein the oligonucleotide probes are in an array. The methodsinclude oligonucleotides probes that comprise a label selected from thegroup consisting of a radioactive label, fluorescent label, enzymaticlabel, avidin label or biotin label.

Alternatively, the invention provides for methods of determining whethera donor tissue intended for transplant or transfusion will likely berejected in a human recipient wherein the human recipient has HNA-3aspecific antibodies, comprising a) obtaining a sample from the tissue,b) extracting nucleic acids from the sample, c) amplifying a fragment ofHNA-3a nucleic acid of SEQ ID NO: 3 from the extracted nucleic acidsusing at least one oligonucleotide primer specific for HNA-3a nucleicacid, and d) detecting the fragment of HNA-3a nucleic acid in thesample, wherein the presence of HNA-3a nucleic acid in the sampleindicates that the sample is likely to be rejected in a human recipientthat has HNA-3a specific antibodies. These methods may be carried outwith primers that amplify a fragment of HNA-3a nucleic acid encodes anamino acid sequence selected from the group consisting of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41 and SEQ ID NO: 42.

The invention also provides for methods of determining whether a donortissue intended for transplant or transfusion is likely to be rejectedin a human recipient wherein the human recipient has HNA-3b specificantibodies, comprising a) obtaining a sample from the tissue, b)extracting nucleic acids from the sample, c) amplifying a fragment ofHNA-3b nucleic acid of SEQ ID NO: 4 from the extracted nucleic acidsusing at least one oligonucleotide primer specific for HNA-3b nucleicacid, and d) detecting the fragment of HNA-3b nucleic acid in thesample, wherein the presence of HNA-3b nucleic acid in the sampleindicates that the sample is likely to be rejected in a human recipientthat has HNA-3b specific antibodies. These methods may be carried outwith at least one primer that amplifies a fragment of HNA-3b nucleicacid that encodes an amino acid sequence selected from the groupconsisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 26.

The invention further provides for methods of determining whether adonor tissue intended for transplant or transfusion will likely berejected in a human recipient wherein the human recipient has HNA-3a orHNA-3b specific antibodies, in addition to detecting the HNA-3a orHNA-3b allele, the method further comprises one or more of the followingsteps: amplifying a fragment of HNA-1 nucleic acid (SEQ ID NO: 5) usingat least one oligonucleotide primer specific for HNA-1 nucleic acid,amplifying a fragment of HNA-2 nucleic acid (SEQ ID NO: 7) using atleast one oligonucleotide primer specific for HNA-2 nucleic acid,amplifying a fragment of HNA-4 nucleic acid (SEQ ID NO: 9) using atleast one oligonucleotide primer specific for HNA-4 nucleic acid,amplifying a fragment of HNA-5 nucleic acid (SEQ ID NO: 11) using atleast one oligonucleotide primer specific for HNA-5 nucleic acid, oramplifying a fragment of HLA nucleic acid using at least oneoligonucleotide primer specific for HLA nucleic acid, and detecting thefragment of any HNA or HLA nucleic acid in the sample, wherein thepresence of an HNA or HLA nucleic acid in the sample indicates that thesample is likely to be rejected in a human recipient.

In another embodiment, the invention provides for methods of determiningthe susceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD wherein the donor tissue contains HNA-3aspecific antibodies, comprising a) obtaining a biological sample fromthe human transplant or transfusion recipient prior to transplantationor transfusion, b) extracting nucleic acids from the biological sample,c) amplifying a fragment of the HNA-3a nucleic acid (SEQ ID NO: 3) fromthe extracted nucleic acids using at least one oligonucleotide primerspecific for HNA-3a nucleic acid in the biological sample, and d)detecting the presence of HNA-3a nucleic acid in the biological sample,wherein the presence of HNA-3a nucleic acid in the biological sampleindicates that the human transfusion or transplant recipient issusceptible for developing TRALI or GVHD. These methods may be carriedout with primers that amplify a fragment of HNA-3a nucleic acid sequencethat encodes an amino acid sequence selected from the group consistingof SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

In another embodiment, the invention provides for methods of determiningthe susceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD, wherein the donor tissue contains anti-HNA-3bantibodies, comprising a) obtaining a biological sample from the humantransplant or transfusion recipient prior to transplantation ortransfusion, b) extracting nucleic acids from the biological sample, c)amplifying a fragment of the HNA-3b nucleic acid (SEQ ID NO: 4) from theextracted nucleic acids using at least one oligonucleotide primerspecific for HNA-3b nucleic acid, and d) detecting the presence ofHNA-3b nucleic acid in the biological sample, wherein the presence ofHNA-3b nucleic acid in the biological sample indicates that the humantransfusion or transplant recipient is susceptible for developing TRALIor GVHD. These methods may be carried out with primers that amplify afragment of nucleic acid sequence that encodes an amino acid sequenceselected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO:26.

In a further embodiment, the invention provides for methods ofdetermining the susceptibility of a human transplant or transfusionrecipient for developing TRALI or GVHD, wherein the donor tissuecontains HNA-3a or HNA-3b specific antibodies, in addition to detectingthe HNA-3a or HNA-3b allele, the method further comprises one or more ofthe following steps: amplifying a fragment of HNA-1 nucleic acid (SEQ IDNO: 5) using at least one oligonucleotide primer specific for HNA-1nucleic acid, amplifying a fragment of HNA-2 nucleic acid (SEQ ID NO: 7)using at least one oligonucleotide primer specific for HNA-2 nucleicacid, amplifying a fragment of HNA-4 nucleic acid (SEQ ID NO: 9) usingat least one oligonucleotide primer specific for HNA-4 nucleic acid,amplifying a fragment of HNA-5 nucleic acid (SEQ ID NO: 11) using anoligonucleotide primer specific for HNA-5 nucleic acid, or amplifying afragment of HLA nucleic acid using at least one oligonucleotide primerspecific for HLA nucleic acid, and detecting the fragment of any one ofHNA-1, HNA-2, HNA-3a, HNA-3b, HNA-4, HNA-5 or HLA nucleic acid in thesample, wherein the presence of any one of HNA-1, HNA-2, HNA-3a, HNA-3b,HNA-4, HNA-5 or HLA nucleic acid in the sample indicates that the sampleis likely to induce TRALI or GVHD in a human recipient.

In any of the preceding methods of the invention, the tissue sample orbiological sample is selected from the group consisting of blood, bloodderivatives, plasma, serum, cells, and tissues. In particular, thetissue sample or biological sample may be a neutrophil.

The invention also provides for kits for carrying out any of theforegoing methods. In particular, the invention provides for kits fordetecting HNA-3a and/or HNA-3b antibodies in conjunction with detectingantibodies specific for one or more of HNA-1, HNA-2, HNA-4, HNA-5 and anHLA. The invention also provides for kits for detecting HNA-3a and/orHNA-3b antigens in conjunction with detecting HNA-1, HNA-2, HNA-4, HNA-5and an HLA. The invention further provides for methods of detecting anHNA-3a or HNA-3b allele in conjunction with detecting an allele for oneor more of HNA-1, HNA-2, HNA-4, HNA-5 and an HLA.

In one embodiment, the invention provides for determining whether adonor tissue intended for transplant or transfusion will induce TRALI orGVHD in a human recipient wherein the human recipient expresses theHNA-3a antigen, wherein the kits comprise a polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereof,and one or more polypeptides or antigenic fragments thereof selectedfrom the group consisting of Fc-γ receptor IIIb polypeptide, CD177polypeptide, CD11b polypeptide, CD11a polypeptide and an HLA antigen.The kit may also comprise a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or an antigenic fragment thereof for detectionof HNA-3b specific antibodies in conjunction with detection of HNA-3aspecific antibodies.

The kit may optionally also comprise an antibody specific for HNA-3a andone or more antibodies that specifically bind to a peptide comprising anantigen selected from the group consisting of HNA-1, HNA-2, HNA-4, HNA-5and HLA. The HNA-3a polypeptide fragment of these kits may comprise anamino acid sequence selected from the group consisting of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41 and SEQ ID NO: 42.

The invention also provides for kits for determining whether a donortissue intended for transplant or transfusion is likely to be rejectedin a human recipient wherein the human recipient has HNA-3a specificantibodies, wherein the kits comprise an antibody specific for HNA-3a,and one or more antibodies that specifically bind to a peptidecomprising an antigen selected from the group consisting of HNA-1,HNA-2, HNA-3b, HNA-4, HNA-5 or HLA.

In addition, the invention provides for kits for determining thesusceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD, wherein the donor tissue contains anti-HNA-3aantibodies, wherein the kits comprise an antibody specific for HNA-3a,and one or more antibodies that specifically bind to a peptidecomprising an antigen selected from the group consisting of HNA-1,HNA-2, HNA-3b, HNA-4, HNA-5 and HLA.

Any of the preceding kits may further comprise a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereofand/or one or more polypeptides or antigenic fragments thereof selectedfrom the group consisting of Fc-γ receptor IIIb polypeptide, CD177polypeptide, CD11b polypeptide, CD11a polypeptide and an HLA antigen.The kit may also comprise an antibody specific for HNA-3b for detectionof HNA-3b antigen in conjunction with detection of HNA-3a antigen.

The invention also provides for kits for determining whether a donortissue intended for transplant or transfusion will induce TRALI or GVHDin a human recipient wherein the human recipient expresses the HNA-3bantigen, wherein the kits comprise a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2 or an antigenic fragment thereof, and oneor more polypeptides or antigenic fragments thereof selected from thegroup consisting of Fc-γ receptor IIIb polypeptide, CD177 polypeptide,CD11b polypeptide, CD11a polypeptide and an HLA antigen. The kit mayalso comprise an antibody specific for HNA-3b and/or one or moreantibodies that specifically bind to a peptide comprising an antigenselected from the group consisting of HNA-1, HNA-2, HNA-3b, HNA-4, HNA-5and HLA. In particular, the invention contemplates kits in which theantigenic fragment of the amino acid of SEQ ID NO: 2 are selected fromthe group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 26.

The invention also provides for kits for determining whether a donortissue intended for transplant or transfusion is likely to be rejectedin a human recipient wherein the human recipient has HNA-3b specificantibodies, wherein the kit comprises an antibody specific for HNA-3b,and one or more antibodies that specifically bind to a peptidecomprising an antigen selected from the group consisting of HNA-1,HNA-2, HNA-3a, HNA-4, HNA-5 and HLA.

In another embodiment, the invention provides for kits for determiningthe susceptibility of a human transplant or transfusion recipient fordeveloping TRALI or GVHD, wherein the donor tissue contains anti-HNA-3bantibodies, wherein the kits comprise an antibody specific for HNA-3b,and one or more antibodies that specifically bind to a peptidecomprising an antigen selected from the group consisting of HNA-1,HNA-2, HNA-3a, HNA-4, HNA-5 and HLA.

These kits may further comprise a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or an antigenic fragment thereof and/or one ormore polypeptides or antigenic fragments thereof selected from the groupconsisting of Fc-γ receptor IIIb polypeptide, CD177 polypeptide, CD11bpolypeptide, CD11a polypeptide and an HLA antigen.

In addition, the preceding kits may comprise a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or an antigenic fragment thereoffor detection of HNA-3a specific antibodies in conjunction withdetection of HNA-3b specific antibodies.

Any of the preceding kits may also comprise a secondary antibody. Theprimary or secondary antibody may comprise a label selected from thegroup consisting of a radioactive label, fluorescent label, enzymaticlabel, avidin label or biotin label.

In a further embodiment, the invention provides for kits for determiningwhether a donor tissue intended for transplant or transfusion is likelyto be rejected in a human recipient wherein the human recipientexpresses HNA-3a specific antibodies, wherein the kits comprise anoligonucleotide probe that hybridizes to a fragment of the nucleic acidsequence of SEQ ID NO: 3, and one or more oligonucleotide probes thathybridizes to a fragment of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, and an HLA nucleotide sequence.

The invention also provides for kits for determining the susceptibilityof a human transplant or transfusion recipient for developing TRALI orGVHD, wherein the donor tissue contains anti-HNA-3a antibodies, whereinthe kits comprise an oligonucleotide probe that hybridizes to a fragmentof the nucleic acid sequence of SEQ ID NO: 3, and one or moreoligonucleotide probes that hybridizes to a fragment of a nucleic acidsequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, and an HLA nucleotide sequence. Theoligonucleotide probes of these kits may comprise a fragment of thenucleotide sequence that encodes an amino acid sequence selected fromthe group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

The invention also provides for kits for determining whether a donortissue intended for transplant or transfusion is likely to be rejectedin a human recipient wherein the human recipient expresses HNA-3bspecific antibodies, wherein the kit comprises an oligonucleotide probethat hybridizes to a fragment of the nucleic acid sequence of SEQ ID NO:4, and one or more oligonucleotide probes that hybridizes to a fragmentof a nucleic acid sequence selected from the group consisting of SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and an HLA nucleotidesequence.

The invention also provides for kits for determining the susceptibilityof a human transplant or transfusion recipient for developing TRALI orGVHD, wherein the donor tissue contains anti-HNA-3b antibodies, whereinthe kits comprise an oligonucleotide probe that hybridizes to a fragmentof the nucleic acid sequence of SEQ ID NO: 4, and one or moreoligonucleotide probes that hybridizes to a fragment of a nucleic acidsequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, and an HLA nucleotide sequence. Theoligonucleotide probes of these kits may comprise a fragment of thenucleotide sequence that encodes an amino acid sequence selected fromthe group consisting of a nucleotide sequence that encodes an amino acidsequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQID NO: 26.

In any of the preceding kits the oligonucleotide probe may comprise alabel selected from the group consisting of a radioactive label,fluorescent label, enzymatic label, avidin label and biotin label. Inaddition, the kits may further comprise buffers for gel loading.

In another embodiment, the invention provides for kits for determiningwhether a donor tissue intended for transplant or transfusion is likelyto be rejected in a human recipient wherein the human recipientexpresses HNA-3a specific antibodies, wherein the kits comprise at leastone oligonucleotide primer for amplifying a fragment of the HNA-3anucleic acid of SEQ ID NO: 3, and one or more oligonucleotide primersfor amplifying a fragment of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, and an HLA nucleotide sequence.

The invention also provides for kits for determining the susceptibilityof a human transplant or transfusion recipient for developing TRALI orGVHD, wherein the donor tissue contains anti-HNA-3a antibodies, whereinthe kits comprise at least one oligonucleotide primers for amplifying afragment of the HNA-3a nucleic acid of SEQ ID NO: 3, and one or moreoligonucleotide primers for amplifying a fragment of a nucleic acidsequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, and an HLA nucleotide sequence. Theseprimers may amplify a fragment of HNA-3a nucleic acid which encodes anamino acid sequence selected from the group consisting of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41 and SEQ ID NO: 42.

The preceding kits may further comprise a fragment of the HNA-3a nucleicacid that is amplified by the oligonucleotide primers and/or one or morefragments of a nucleic acid sequence selected from the group consistingof SEQ ID NO: 5, SEQ NO: ID: 7, SEQ ID NO: 9, SEQ ID NO: 11, and an HLAnucleotide sequence that is amplified by the oligonucleotide primers. Inaddition, the kits may further comprise oligonucleotide primers foramplifying a fragment of the HNA-3b nucleic acid of SEQ ID NO: 4 inconjunction with the amplification of a fragment of HNA-3a.

In another embodiment, the invention provides for kits for determiningwhether a donor tissue intended for transplant or transfusion is likelyto be rejected in a human recipient wherein the human recipientexpresses HNA-3b specific antibodies, wherein the kit comprises at leastone oligonucleotide primer for amplifying a fragment of the HNA-3bnucleic acid of SEQ ID NO: 4, and one or more oligonucleotide primersfor amplifying a fragment of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, and an HLA nucleotide sequence.

The invention also provides for kits for determining the susceptibilityof a human transplant or transfusion recipient for developing TRALI orGVHD, wherein the donor tissue contains anti-HNA-3b antibodies, whereinthe kit comprises at least one oligonucleotide primer for amplifying afragment of the HNA-3b nucleic acid of SEQ ID NO: 4, and at least oneoligonucleotide primer for amplifying a fragment of a nucleic acidsequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, and an HLA nucleotide sequence. Theseprimers may amplify a fragment of HNA-3a nucleic acid which encodes anamino acid sequence selected from the group consisting of SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24 and SEQ ID NO: 26.

Any of the preceding kits may also comprise buffers for PCRamplification, dNTP's and buffers for gel loading.

In another embodiment, the invention provides for an isolated HNA-3bpolypeptides such as isolated polypeptides comprising the amino acidsequence SEQ ID NO: 2, isolated polypeptide comprising a fragment of thepolypeptide of SEQ ID NO: 2 wherein the fragment is at least 7 aminoacids in length, at least 10 amino acids in length, at least 20 aminoacids in length or at least 50 amino acids in length. The fragments ofHNA-3b include a fragment comprising amino acid residue 154 of SEQ IDNO: 2, wherein residue 154 is glutamine (Gln). The fragments of HNA-3bpolypeptide include fragments comprising the amino acid sequence of SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 24 and SEQ ID NO: 26 and reacts with or specifically binds toHNA-3b-specific alloantibodies. The invention also provides ofpolynucleotides encoding the HNA-3b polypeptides.

The invention provides for fragments of the HNA-3a polypeptide of SEQ IDNO: 1, such as an isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, wherein thepolypeptide reacts with or specifically binds to HNA-3a-specificalloantibodies. The invention also provides for polynucleotides encodingthese HNA-3a polypeptide fragments.

In another embodiment, the invention provides for use of a HNA-3apolypeptide or fragment thereof for identification of HNA-3a-specificalloantibodies. Use of a HNA-3b polypeptide or fragment thereof foridentification of HNA-3b-specific alloantibodies is also provided.

The invention further provides for a use of a polynucleotide encoding anHNA-3a polypeptide or a fragment thereof which encodes a protein thatreacts with or specifically binds to HNA-3a-specific alloantibodies, fordetermination of the HNA-3a genotype. In addition, the inventionprovides for use of a HNA-3b polynucleotide or a fragment thereof fordetermination of the HNA-3b genotype. For example, the inventionprovides for use of a fragment of SEQ ID NO: 4 comprising codon 154 fordetermination of the HNA-3b genotype.

In a further embodiment, the invention provides for use of a proteincomprising an amino acid sequence of SEQ ID NO: 1 or a fragment thereofwhich reacts with HNA-3a-specific alloantibodies, or a HNA-3a proteinfragment in the analysis of blood samples or blood plasma foridentification of antibodies against the HNA-3a antigen.

The invention also provides for use of a HNA-3b polypeptide in theanalysis of blood samples or blood plasma for identification ofantibodies against the HNA-3b antigen.

Use of a polypeptide comprising an amino acid sequence of SEQ ID NO: 1(HNA-3a) or a fragment thereof or an amino acid of SEQ ID NO: 2 (HNA-3b)or a fragment thereof in a process, which uses the protein or theprotein fragment to separate antibodies from blood samples or bloodplasma.

Use of a polypeptide comprising an amino acid sequence of SEQ ID NO: 1(HNA-3a) or a fragment thereof or a polypeptide of SEQ ID NO: 2 (HNA-3b)or a fragment thereof in a process, which uses the protein or theprotein fragment to produce antibodies, preferably monoclonalantibodies. In a further embodiment, the invention provides for methodsof screening for HNA-3b genotype in a human subject comprising a)obtaining a biological sample from the human subject, b) extractingnucleic acids from the biological sample, and b) detecting a fragment ofa nucleic acid sequence of SEQ ID NO: 4 in the biological sample,wherein the fragment comprises codon 154 of SEQ ID NO: 4, wherein thedetection of codon 154 of SEQ ID NO: 4 indicates that the human subjecthas the HNA-3b genotype. The detecting step may include contacting thenucleic acid with an oligonucleotide probe that hybridizes to a fragmentof the nucleic acid sequence of SEQ ID NO: 4 or amplifying a fragment ofSEQ ID NO: 4 from the extracted nucleic acid using at least oneoligonucleotide primer specific for a fragment of SEQ ID NO: 4. For themethod of screening for HNA-3b genotype, the biological sample that isused may be selected from the group consisting of blood, bloodderivatives, plasma, serum, cells, and tissues.

Sequences of the Invention

SEQ ID NO: 1—human HNA-3a protein

SEQ ID NO: 2—human HNA-3b protein

SEQ ID NO: 3—human HNA-3a DNA

SEQ ID NO: 4—human HNA-3b DNA

SEQ ID NO: 5—human FCγ receptor IIIb DNA (HNA-1)

SEQ ID NO: 6—human FCγ receptor IIIb protein (HNA-1)

SEQ ID NO: 7—human CD177 DNA (HNA-2)

SEQ ID NO: 8—human CD177 protein (HNA-2)

SEQ ID NO: 9—human CD11b DNA (HNA-4)

SEQ ID NO: 10—human CD11b protein (HNA-4)

SEQ ID NO: 11—human CD11a DNA (HNA-5)

SEQ ID NO: 12—human CD11a protein (HNA-5)

SEQ ID NO: 13—amino acids 1-231 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 14—amino acids 55-183 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 15—amino acids 55-164 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 16—amino acids 114-164 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 17—amino acids 55-706 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 18—amino acids 114-706 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 19—amino acids 1-231 of HNA-3b (SEQ ID NO: 2)

SEQ ID NO: 20—amino acids 55-183 of HNA-3b (SEQ ID NO: 2)

SEQ ID NO: 21—amino acids 55-164 of HNA-3b (SEQ ID NO: 2)

SEQ ID NO: 22—amino acids 114-164 of HNA-3b (SEQ ID NO: 2)

SEQ ID NO: 23—amino acids 55-706 of HNA-3b (SEQ ID NO: 2)

SEQ ID NO: 24—amino acids 114-706 of HNA-3B (SEQ ID NO: 2)

SEQ ID NO: 25—amino acids 154-164 of HNA-3a (SEQ ID NO: 1)

SEQ ID NO: 26—amino acids 154-164 of HNA-3b (SEQ ID NO: 2)

SEQ ID NOS: 27-47—fragments of HNA-3a in Table 1

SEQ ID NO: 48—amino acids 145-167 of HNA-3a (SEQ ID NO: 1)

SEQ ID NOS: 49-55—primer sequences

DETAILED DESCRIPTION

The present invention is based on the discovery that HNA-3 is located onthe CTL2 transmembrane protein. A serum sample that was known to induceTRALI in transfusion recipients, but was negative for HNA-1 and HNA-2antibodies, was used to identify the source of HNA-3a antigen. HNA-3awas identified by first comparing the HNA-3a positive and negative cellsurface proteins that were immunoprecipitated by HNA-3a sera. The cellswere characterized with HNA-3a positive and negative sera by flowcytometry. The HNA positive and negative granulocytes were thenincubated with HNA-3a positive sera and the cell surface proteins thatreacted with the sera were immunoprecipitated with Protein-G coatedmagnetic beads as described in detail in Example 6. The protein profileswere first analyzed using SDS-PAGE and two identified proteins having amolecular weight of about 80-100 kD were only present in the positivecells, not in the negative cells. Those proteins were excised from theSDS gel and further analyzed with mass spectrometry (MS) analysis andconfirmed by sequencing the amino acid sequence.

The invention provides for methods of screening biological samples todetect antibodies specific for HNA-3a or HNA-3b antigen. The biologicalsample includes whole blood, blood derivatives, red blood cellconcentrates, plasma, serum, fresh frozen plasma, whole blood derivedplatelet concentrates, apheresis platelets, pooled platelets,intravenous gamma-globulin, cryoprecipitate, cerebrospinal fluid,tissues and cells such as stem cells, neutrophils and granulocytes. Thebiological samples may be obtained from a human donor of tissue or cellsintended for transplantation or a human donor of blood or bloodderivatives intended for transfusion. The biological samples may beobtained from tissues or cells that are intended for transplantation ina human recipient. In addition, the biological sample may be obtainedfrom blood or blood derivatives that are intended for transfusion in ahuman recipient. The biological sample may also be obtained from a humansubject that is an intended recipient of a transplant or transfusion.

The invention also relates to screening for susceptibility ordetermining if the recipient will develop graft versus host disease(GVHD). GVHD is when a tissue comprises immunologically competent cellsor antibodies that attack the recipient. The leading cause of GVHD ishematopoietic cell transplantation, both allogeneic (between 2individuals) and autologous (from the same individual). Solid organtransplants, blood transfusions, and maternal-fetal transfusions alsoreportedly cause GVHD. Acute symptoms of GVDH include abdominal pain ordamage, diarrhea, fever, jaundice, skin rash, vomiting and weight loss.Chronic symptoms of GVHD include dry eyes, dry mouth, hepatitis, lungand digestive tract disorders and skin rash.

The invention also relates to methods and products related todetermining if a transplanted or transfused tissue is likely to berejected by the recipient. Symptoms of rejection include indicationsthat the transplanted organ does not function properly, generaldiscomfort, uneasiness, or ill feeling, pain or swelling in the locationof the organ and fever.

Identification of HNA-3a and HNA-3b

It was therefore an object of the present invention to identify andprovide the protein and DNA sequences of the human neutrophil antigen-3aor -3b (HNA-3a, HNA-3b) involved in TRALI.

In other words, the problem was solved by the provision of the protein(HNA-3a antigen), consisting of an amino acid sequence SEQ ID NO: 1,which reacts with alloantibodies which are specific for HNA-3a, and bythe provision of the protein (HNA-3b antigen), consisting of an aminoacid sequence SEQ ID NO: 2, which reacts with alloantibodies which arespecific for HNA-3b.

Subjects coming into question were investigated for the identificationof HNA-3a-positive and -negative subjects, whose cells can be used forfurther investigations. Antibodies from donors whose blood products havetriggered TRALI, and the white blood cells on the surfaces of which thecorresponding antigens are expressed were used. After the identificationof subjects with high-titer HNA-3a antibodies, with which the antigencan be precipitated, the donors were subjected to a plasmapheresis, inorder to obtain sufficient material for the antigen-antibody reactions.The antigens were precipitated by means of the antibodies and the stillunknown protein/gene structures of HNA-3a were characterized.

In detail, firstly an optimization of the preparation of granulocyteswas developed. Then, by means of a screening program, HNA-3a-positiveand -negative subjects were determined, whose cells were able to be usedfor further investigations. Subjects were then selected with high-titerHNA-3a antibodies, with which the antigen was able to be precipitated. Aplasmapheresis of the selected subjects was carried out in order toobtain sufficient material for the antigen-antibody reactions. Themethod for the preparation of granulocyte membrane proteins for aquantitative gel electrophoresis was first developed with platelets,because these have no nucleus and the preparation was thereforesimplified. The method was then transferred and adapted toleucocytes/granulocytes containing a nucleus. This made possible anoptimization of the preparation of granulocyte membrane proteins. Thecorresponding proteins were analyzed by means of analytical methods. Theenrichment/isolation of HNA-3a from the prepared membrane proteins wascarried out by means of immunoprecipitation, and confirmed by Westernblotting. The protein which carries HNA-3a was identified by massspectrometry; and subsequently, the primary sequence of HNA-3a wasidentified by sequence analysis (SEQ ID NO: 1).

The invention therefore concerns a protein (HNA-3a antigen), consistingof an amino acid sequence SEQ ID NO: 1, which reacts with alloantibodieswhich are HNA-3a-specific. Additionally included are also proteins(HNA-3a antigens) consisting of an amino acid sequence SEQ ID NO: 1, inwhich one or more amino acids have been removed, added or replaced andwhich react with alloantibodies which are HNA-3a specific. Theidentified HNA-3a antigen proved to be a variant of the transmembranereceptor CTL2. This has a molecular weight in the range 80 to 100 kDa,and deglycosylation shifted the band to 64 kDa in the Western blot.

The HNA-3 antigen is expressed on the CTL2 protein on granulocytes andlymphocytes. A single nucleotide polymorphism (SNP) on an extracellularloop is crucial (SNP rs2288904), as this SNP is the difference betweenHNA-3a and HNA-3b. This SNP allows for genotyping of blood donors inrelation to their HNA-3a/HNA-3b status. The polynucleotide encodingHNA-3a has a “G” (guanine) at position 461 and as a result encodes an“R” (Arginine, Arg) at position 154 of the HNA-3a amino acid sequence,and therefore represents the HNA-3a allele. The polynucleotide encodingHNA-3b has an “A” (adenine) at position 461 and as a result encodes a“Q” (glutamine, Gln) at position 154 of the amino acid sequence, andtherefore represents the HNA-3b allele.

The primary sequence of the HNA-3b antigen was determined through aminoacid exchange at position 154 arginine (Arg, R) to glutamine (Gln, Q)(see SEQ ID NO: 2). Accordingly, the invention concerns a protein(HNA-3b antigen) consisting of an amino acid sequence of SEQ ID NO: 2,which reacts with HNA-3b specific alloantibodies Likewise, the inventionincludes HNA-3b proteins consisting of an amino acid sequence of SEQ IDNO: 2, in which one or more amino acids have been removed, added orreplaced and which react with HNA-3b specific alloantibodies.

Likewise, the invention provides for proteins, both with respect to theHNA-3a and also to the HNA-3b, which are protein fragments with a chainlength of at least 7, at least 10 amino acids, at least 20 amino acids,or at least 50 amino acids.

Subsequently, the HNA-3a gene was isolated and heterologously expressed.The corresponding DNA sequences for HNA-3a correspond to the sequenceset out as the nucleotide sequence SEQ ID NO: 3 and all sequenceshybridizing therewith, which codes an HNA-3a antigen described above,which reacts with or binds to HNA-3a specific alloantibodies. Theinvention also includes a nucleotide sequence which has an identity onthe nucleotide level of at least 90%, preferably at least 95%, mostpreferably at least 98% to SEQ ID NO: 3 and all sequences hybridizingtherewith, which codes an HNA-3a antigen described above, and whichreacts with or binds to HNA-3a specific alloantibodies.

Additionally, the invention provides for splice variants of a nucleotidesequence SEQ ID NO: 3, which are at least 70% identical to thenucleotide sequence SEQ ID NO: 3. Preferably, the sequence identity isat least 80%, more preferably 90% and most preferably 95% to SEQ ID NO:3.

The invention further provides for a nucleotide sequence SEQ ID NO: 4and all sequences hybridizing therewith, which codes the HNA-3b antigendescribed above, which reacts with HNA-3b specific alloantibodies. Thisalso includes a nucleotide sequence which has an identity on thenucleotide level of at least 90%, preferably at least 95%, mostpreferably at least 98% and all sequences hybridizing therewith whichcodes a HNA-3b antigen described above, and which reacts withalloantibodies which are HNA-3b specific. Exemplary stringenthybridization conditions comprise hybridization at 65° C. and washingthree times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. Additionalexemplary stringent hybridization conditions comprise hybridization in0.02 M to 0.15 M NaCl at temperatures of about 50° C. to 70° C. or0.5×SSC 0.25% SDS at 65° for 15 minutes, followed by a wash at 65° C.for a half hour or hybridization at 65° C. for 14 hours followed by 3washings with 0.5×SSC, 1% SDS at 65° C.

Additionally, the invention provides for splice variants of a nucleotidesequence of SEQ ID NO: 4, which are at least 70% identical to thenucleotide sequence SEQ ID NO: 4. Preferably, the sequence identity isat least 80%, more preferably 90% and most preferably 95%.

Sequence identity or identity on the nucleotide level generally means100% identity.

Based on the determined primary structures, the method for recombinantproduction of the antigen was optimized on the basis of the alreadyknown proteins/antigens HNA-1a, -1b, -1c, -2a. The results obtained forthe HNA-1 and HNA-2 antigens were transferred to HNA-3a or HNA-3b, sothat these antigens would be produced in a suitable expression systemsuch as expression in Escherichia coli, in eukaryotic cells, in insectcells.

The present invention accordingly contains the use of a protein (HNA-3aantigen) consisting of an amino acid sequence of SEQ ID NO: 1 for theidentification of HNA-3a specific alloantibodies. Likewise included inthe invention is the use of a protein (HNA-3b antigen) consisting of anamino acid sequence of SEQ ID NO: 2 for the identification of HNA-3bspecific alloantibodies.

In addition, the present invention includes the use of a nucleotidesequence of SEQ ID NO: 3 for determining the HNA-3a genotype, and theuse of a nucleotide sequence of SEQ ID NO: 4 for determining the HNA-3bgenotype.

The methods of the present invention may be carried out using ELISAassays, flow cytometry, immunofluorescence methods, electro-chip assays,PCRs and agglutination tests.

Likewise, the invention provides for a test system for determiningHNA-3a specific alloantibodies, which bind to a protein (HNA-3a antigen)consisting of an amino acid sequence of SEQ ID NO: 1. The invention alsoprovides for a test system for determining HNA-3b specificalloantibodies, which bind to a protein (HNA-3b antigen) consisting ofan amino acid sequence of SEQ ID NO: 2.

The invention also provides for a test system for determining the HNA-3agenotype, which comprises a nucleotide sequence of SEQ ID NO: 3 and fora test system for determining the HNA-3b genotype which comprises anucleotide sequence of SEQ ID NO: 4.

According to the invention, the protein (HNA-3a antigen), consisting ofan amino acid sequence of SEQ ID NO: 1, is used in the analysis of bloodsamples or blood plasma for the identification of antibodies thatspecifically bind to HNA-3a antigen. Likewise, the protein (HNA-3bantigen), consisting of an amino acid sequence SEQ ID NO: 2 is used inthe analysis of blood samples or blood plasma for the identification ofantibodies against the HNA-3b antigen.

The invention further comprises the use of a protein (HNA-3a antigen)consisting of an amino acid sequence of SEQ ID NO: 1 in a method whichuses the antigen in order to separate antibodies from blood samples orblood plasma. The invention likewise comprises the use of a protein(HNA-3b antigen) consisting of an amino acid sequence of SEQ ID NO: 2 asdescribed above in a method which uses the antigen in order to separateantibodies from blood samples or blood plasma. Particularly preferred isthe use of the protein in adsorption methods such as plasmapheresis.

The invention further provides for the use of a protein (HNA-3a antigen)consisting of an amino acid sequence of SEQ ID NO: 1 in a method whichuses the antigen in order to produce antibodies, preferably monoclonalantibodies. Likewise the invention provides for the use of a protein(HNA-3b antigen) consisting of an amino acid sequence of SEQ ID NO: 2 ina method which uses the antigen in order to produce antibodies,preferably monoclonal antibodies.

Antigenic Fragments of HNA-3a

Epitope mapping of antigenic fragments of HNA-3a or HNA-3b, thatgenerate HNA-3a or HNA-3b specific antibodies, may be identified usingmethods standard in the art such as site-specific mutagenesis, geneticengineering, analysis of CTL2 peptide libraries, predictive algorithms,functional assays, such as ELISpot or intracellular cytokine staining,and cellular binding assays. High throughput systems for analysis ofpeptide libraries are commercially available, such as the REVEAL &ProVE™ System (Proimmune, Springfield. VA).

Preferred protein fragments of HNA-3a (SEQ ID NO: 1) include at leastthe amino acid sequence of SEQ ID NO: 13 (amino acids 1-231 of SEQ IDNO: 1), at least the amino acid sequence of SEQ ID NO: 14 (amino acids55-183 of SEQ ID NO: 1), at least the amino acid sequence of SEQ ID NO:15 (amino acids 55-164 of SEQ ID NO: 1), at least the amino acidsequence of SEQ ID NO: 16 (amino acids 114-164 of SEQ ID NO: 1), atleast the amino acid sequence of SEQ ID NO: 25 (amino acids 154-164 ofSEQ ID NO: 1), at least the amino acid sequence of SEQ ID NO: 17 (aminoacids 55-706 of SEQ ID NO: 1), and at least the amino acid sequence ofSEQ ID NO: 18 (amino acids 114-706 of SEQ ID NO: 1), which would reactwith or bind to HNA-3a-specific antibodies such as alloantibodies.

Furthermore, the invention relates to any protein fragment consisting ofamino acid sequence of SEQ ID NO: 1 as described herein in which one ormore amino acids has been removed, added or have been replaced and whichreacts with or binds to HNA-3a-specific antibodies or alloantibodies.

The present invention relates to protein fragments of the amino acidsequence of SEQ ID NO: 2. Preferred protein fragments of HNA-3b (SEQ IDNO: 2) include at least the amino acid sequence of SEQ ID NO: 19 (aminoacids 1-231 of SEQ ID NO: 2), at least the amino acid sequence of SEQ IDNO: 20 (amino acids 55-183 of SEQ ID NO: 2), at least the amino acidsequence of SEQ ID NO: 21 (amino acids 55-164 of SEQ ID NO: 2), at leastthe amino acid sequence of the SEQ ID NO: 22 (amino acids 114-164 of SEQID NO: 2), at least the amino acids of SEQ ID NO: 26 (amino acids154-164 of SEQ ID NO: 2), at least the amino acids of SEQ ID NO: 23(amino acids 55-706 of SEQ ID NO: 2) and at least the amino acids of SEQID NO: 24 (amino acids 114-706 of SEQ ID NO: 2), which react with orbind to the HNA-3b specific antibodies such as alloantibodies.

Likewise, the invention relates to protein fragments consisting of anamino acid sequence of SEQ ID NO: 2, as described above, in which one ormore amino acids has been removed, added or replaced and which reactswith or binds to the HNA-3b specific antibodies or alloantibodies.

Method of Detecting HNA-3 Specific Antibodies

The invention provides for methods of detecting HNA-3a or HNA-3bspecific antibodies in a biological sample. The invention alsocontemplates detecting other antibodies such as antibodies specific forHNA-1, HNA-2 or HLA in combination with detecting HNA-3a or HNA-3bspecific antibodies. Methods of detecting of antibody includenon-specific and specific assays such as, granulocyte immunofluorescencetest, granulocyte immunofluorescence flow cytometry assay (GIFT-FC),monoclonal antibody immobilization of granulocyte antigens (MAIGA)assay, single radial immunodiffussion assay (SRID), enzyme immunoassayand hemagglutination inhibition assay (HAI).

An exemplary non-specific assay uses intact granulocytes as a target,e.g. GIFT-FC uses a panel of neutrophils with different HNAs (Davoren,et al. Transfusion 43(5): 641-5, 2003, Kobayashi et al, Ped. Res. 26:246-249). The neutrophils are first incubated with test sera followed byincubation with a fluorescently labeled secondary antibody, such asantihuman polyvalent immunoglobulin, IgG, IgM and IgA. After washing,the antibody binding to the cell suspensions is examined by flowcytometry.

An exemplary specific assay uses immobilized HNA glycoprotein as atarget, e.g. MAIGA assay. MAIGA is an ELISA-based test that uses HNA-3specific monoclonal antibodies to capture the neutrophil antigens withintest sera. Subsequently, the cell mixtures are incubated with an enzymelabeled secondary antibody, such as anti-mouse IgG, and binding isdetected with a colorimetric assay (Bux, et al. Transfusion Med. 3(2):157-62, 1993, Metcalfe &Waters, Transfusion Med. 2:283-287, 1992.)

ELISA assay is used to determine total antibodies in the sample. Theimmunogen, e.g. the HNA-3a polypeptide of SEQ ID NO: 1, the HNA-3bpolypeptide of SEQ ID NO: 2 or antigenic fragments thereof, is adsorbedto the surface of a microtiter plate. The test serum is exposed to theplate followed by an enzyme linked immunoglobulin, such as IgG. Theenzyme activity adherent to the plate is quantified by any convenientmeans such as spectrophotometers and is proportional to theconcentration of antibody directed against the immunogen present in thetest sample. In addition, HNA-3a or HNA-3b polypeptide or antigenicfragments thereof may be attached to solid substrates such as membranes,beads, filters, glass, silicon, metal, metal-alloy, anopore, polymeric,nylon or plastic for detection of antibodies specific for HNA-3a orHNA-3b.

The SRID assay utilizes a layer of a gel, such as agarose, containingthe antigen being tested. A well is cut in the gel and the test sera areplaced in the well. Diffusion of the antibody out into the gel leads tothe formation of a precipitation ring whose area is proportional to theconcentration of the antibody in the serum being tested.

HAI utilizes the capability of an immunogen to agglutinate chicken redblood cells (or the like). The assay detects neutralizing antibodies,i.e., those antibodies able to inhibit hemagglutination. Dilutions ofthe test serum are incubated with a standard concentration of immunogen,followed by the addition of the red blood cells. The presence ofneutralizing antibodies will inhibit the agglutination of the red bloodcells by the immunogen.

Additional assays to detect circulating anti-HNA-3a or anti-HNA-3bantibody in the serum of the transplant or transfusion patient may beused. In such an assay, serum is screened for the presence ofanti-HNA-3a or HNA-3b antibodies through detection ofcomplement-mediated lytic activity. Serum is screened forcomplement-mediated lytic activity against T and B lymphocytes from apanel of individuals representing the most frequently encountered HNA-3aor HNA-3b antigens. The assay is performed in the presence or absence ofdithioerythritol.

The methods of detecting an HNA-3a or HNA-3b antibodies of the inventionmay be carried out with neutrophils or any cell type transformed ortransfected to express HNA-3a or HNA-3b. The methods may be carried outwill cells that do not endogenously express HNA-3a or HNA-3, such asB-cells, CHO cells or insect cells. The invention also contemplatesusing cells that express low levels of HNA-3a or HNA-3b and increasingexpression of the endogenous HNA-3a or HNA-3b protein by insertingheterologous promoters or enhancers, or increasing copy number of theHNA-3a or HNA-3b gene.

Exemplary B cell that may be used in the methods of the inventioninclude EB-3 cells (ATCC CCL85), K-562 cells (ATCC CCL243), RAJI cells(ATCC CCL86), Jiyoye cells (CCL87), IM-9 (ATCC159), Daudi cells (ATCCCCL213), NC-37 cells (ATCC 214), Mo—B cells (ATCC 245), KG-1 cells (ATCCCCL246), H2126 cells (ATCC 256), BL2126 cells (ATCC 256) and MCL-5 cells(ATCC CCL10575). Other exemplary cells that may be used in the methodsof the invention include Chinese hamster ovary cells (CHO) (ATCC No.CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA,97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells(ATCC No. CRL1573), or 3T3 cells (ATCC No. CCL92), monkey COS-1 (ATCCNo. CRL1650) and COS-7 cell (ATCC No. CRL1651), and CV-1 cells (ATCC No.CCL70). In addition, insect cells may be used in the methods of theinvention such as SF-9 and HI5 cells.

Furthermore, cells that endogenously express HNA-3a or HNA-3b at low ormoderate levels may be modified to enhance or overexpress endogenousHNA-3a or HNA-3b. For example a promoter, enhancer element, or anexogenous transcription modulatory element is inserted in the genome ofthe intended cell in proximity and orientation sufficient to influencethe transcription of DNA encoding the HNA-3a or HNA-3b polypeptide. Thecontrol element controls a portion of the DNA present in the host cellgenome. Thus, the expression of the HNA-3a or HNA-3b polypeptide may beachieved not by transfection of DNA that encodes the HNA-3a or HNA-3bgene itself, but rather by the use of targeting DNA (containing regionsof homology with the endogenous gene of interest) coupled with DNAregulatory segments that provide the endogenous gene sequence withrecognizable signals for transcription.

The invention also provides for methods of detecting HNA-3a or HNA-3bspecific antibodies within a biological sample by contacting abiological sample with an aptamer that mimics an HNA-3a or HNA-3bantigenic fragment or epitope. Aptamers are macromolecules comprisingsingle stranded oligonucleotides that have a sequence-dependentthree-dimensional shape that will bind a target protein with highaffinity and specificity. The invention contemplates developing andusing aptamers that have a sequence that mimics the HNA-3a or HNA-3bepitope and therefore binds to HNA-3a or HNA-3b specific antibodies.These aptamers may be used in any of the methods of the invention todetect the presence of HNA-3a or HNA-3b specific antibodies.

The aptamers of the invention may comprise single stranded RNA or DNAoligonucleotides ranging in size between 15 and 50 bases that are fusedto a scaffold such as thioredoxin. The aptamers will mimic the physicalor structural characteristics of the HNA-3a and HNA-3b peptides of theinvention. The aptamers are generally derived from combinatoriallibraries through an in vitro selection process known as SystematicEvolution of Ligands through Exponential enrichment (SELEX). Exemplarymethods for identifying and synthesizing aptamers against HNA-3a orHNA-3b antibodies are presented in Lo, Antibody Engineering: methods andprotocols Vol 248 of Methods in Molecular Biology, Humana Press 2004,Klussmann, The Aptamer Handbook: functional oligonucleotides and theirapplications Wily-VCH, 2006, and Jayasena Clin. Chem. 45:168-1650, 1999.Any of the assays described herein may be used to confirm that thecontemplated aptamers bind to HNA-3a or HNA-3b specific antibodies.

Furthermore, the invention provides for methods of detecting HNA-3a orHNA-3b specific antibodies using peptides that mimic the secondary ortertiary structure of the antigenic fragments of HNA-3a or HNA-3b, whilediffering in primary amino acid structure. The structuralcharacteristics of these peptides cause the HNA-3a or HNA-3b antibodiesto cross react with these peptides. These peptides may be identifiedusing standard methods in the art such as phage display peptidelibraries and combinatorial libraries.

Methods of Distinguishing HNA-3a or HNA-3b Specific Antibodies

Any of the techniques described herein to detect HNA-3a or HNA-3bspecific antibodies in a biological sample also may be used todistinguish if a particular antibody specifically binds to HNA-3a orHNA-3b. The assays would be carried out with the full length polypeptideor peptides that comprise amino acid 154. In particular, the peptidesused in these assays may retain any secondary or tertiary structure thatwill distinguish the HNA-3a and HNA-3b epitopes.

Furthermore, assays using cells or tissues known to express HNA-3a orHNA-3b may be used to identify and distinguish HNA-3a or HNA-3b specificantibodies. These assays would include cells transfected or transformedto express HNA-3a or HNA-3b.

Isolation of these HNA-3a or HNA-3b specific antibodies is useful forcarrying out the methods of the invention. In addition, the kits of theinvention may comprise isolated HNA-3a or HNA-3b specific antibodies.

Methods of Detecting HNA-3 Protein

The invention provides for methods of detecting HNA-3a or HNA-3b in abiological sample. The term “HNA-3a” refers to the full length sequenceof SEQ ID NO: 1 or at least a fragment of the amino acid sequence of SEQID NO: 1. The term “HNA-3b” refers to the full length amino acidsequence of SEQ ID NO: 2 or is at least a fragment of the amino acidsequence of SEQ ID NO: 2. Antigenic fragments of HNA-3a or HNA-3b thatcomprise particular epitopes that generate specific antibodies are ofinterest. For example, regions of the amino acid sequence of SEQ ID NO:1 or 2 that are exposed to the cell surface are more likely to comprisean epitope.

Exemplary antigenic fragments that may be used to generate HNA-3aspecific antibodies include the amino acid sequence of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41 and SEQ ID NO: 42. Exemplary antigenic fragments that may beused to generate HNA-3b specific antibodies include the amino acidsequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 26.

The primary structure of CTL2 indicates the existence of multiplepolymorphic regions in both the coding region and promoter region of theCTL2 gene. Polymorphisms in the CTL2 gene may provide polymorphicinformation for HNA-3a or HNA-3b. The differences within the promoterregion may cause different transcription efficiency and thus influencethe expression of the CTL2 polypeptide. The polymorphisms in the codingregion can alter the CTL2 protein conformation and thus the differentpolymorphic CTL2 proteins become immunogenic to each other. For example,nucleotide 461 of HNA-3a/HNA-3b is polymorphic wherein the HNA-3a alleleis a “G” that encodes an arginine at position 154 and the HNA-3b alleleis an “A” and encodes a glutamine.

The human neutrophil antigen HNA-1 has polymorphic epitopes. HNA-1 hasthree alleles, HNA-1a, HNA-1b and HNA-1c, which are the result ofpolymorphisms within the FcγRIIIb gene. HNA-1a and HNA-1b differ by fouramino acids. The HNA-1c differs from HNA-1b by a single nucleotidesubstitution (C-to-A) at nucleotide 266 that results in a change ofalanine to aspartate at amino acid 78. As described above, thepolymorphisms in the CTL2 gene may result in HNA-3 polymorphic epitopessimilar to those observed for HNA-1.

However, the human neutrophil antigen HNA-2 has a monomorphic epitope,in which a portion of the population does not express HNA-2. HNA-2 hasonly one well-described allele, HNA-2a. HNA-2a deficiency is caused by atranscription defect which exists among 5-10% individuals. Thoseindividuals may generate HNA-2a antibody when exposed to the HNA-2aantigen. Therefore, it is contemplated that HNA-3A or HNA-3b may have amonomorphic epitope similar to HNA-2.

The invention also contemplates detecting additional antigens, such asHNA-1, HNA-2, HNA-4, HNA-5 and/or HLA, in combination with detectingHNA-3a or HNA-3b in a biological sample.

Commercial antibodies that bind to human CTL2 may be used in the methodsof the invention. Exemplary commercial antibodies include humanmonoclonal anti-SLC44A2 antibody (clone 3D11) and human anti-SLC44A2polyclonal antibody both available from Sigma Aldrich (St. Louis, Mo.).Additional exemplary antibodies include SLC44A2 antibody (ab57570)available from Abcam (Cambridge, Mass.), CTL2 monoclonal antibody (M01),clone 3D11 available from Abnova (Walnut, Calif.), Mouse Polyclonalanti-SLC44A2-solute carrier family 44, member 2, MaxPab Antibody andMouse polyclonal anti-CTL2 available from Novus Biologicals (Littleton,Colo.).

The antibodies of the invention may be polyclonal antibodies, monoclonalantibodies, antibody fragments which retain their ability to bind theirunique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chainantibodies and human or humanized antibodies. Antibodies may begenerated by techniques standard in the art using the HNA-3a or HNA-3bepitope on CTL2 or antigenic fragments of SEQ ID NO: 1 or SEQ ID NO: 2.Antibody molecules of the present invention include the classes of IgG(as well as subtypes IgG 1, IgG 2a, and IgG2b), IgM, IgA, IgD, and IgE.

The antibodies of the invention may be labeled for detection of bindingwithin the biological sample. The antibodies may comprise a radioactivelabel such as ³H, ¹⁴C, ³²P, 35S, or 125I. In addition, the labels may bea fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, phycoerythrin, rhodamine, or luciferin. The labels maybe enzymes such as alkaline phosphatase, β-galactosidase, biotin andavidin or horseradish peroxidase (Bayer et al., Meth. Enz., 184:138-163(1990)).

The HNA-3a and HNA-3b specific antibodies may be attached to solidsubstrates such as membranes, beads, filters, glass, silicon, metal,metal-alloy, anopore, polymeric, nylon or plastic for detection ofHNA-3a or HNA-3b in a biological sample.

Antigens of the invention may be a whole protein, a truncated protein, afragment of a protein or a peptide. Antigens may be naturally occurring,genetically engineered variants of the protein, or may be codonoptimized for expression in a particular mammalian subject or host.Generally, a B-cell epitope will include at least about 5 amino acidsbut can be as small as 3-4 amino acids.

Normally, an epitope will include between about 7 and 15 amino acids,such as, 9, 10, 12 or 15 amino acids. The term “antigen” denotes bothsubunit antigens, (i.e., antigens which are separate and discrete from awhole organism with which the antigen is associated in nature).Antibodies such as anti-idiotype antibodies, or fragments thereof, andsynthetic peptide mimotopes, that are synthetic peptides which can mimican antigen or antigenic determinant, are also captured under thedefinition of antigen as used herein.

Furthermore, for purposes of the present invention, an “antigen” refersto a protein, which includes modifications, such as deletions, additionsand substitutions, generally conservative in nature, to the naturallyoccurring sequence, so long as the protein maintains the ability toelicit an immunological response, as defined herein. These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutations of hosts which produce theantigens. Antigens of the present invention may also be codon optimizedby methods known in the art to improve their expression orimmunogenicity in the host.

Specific binding of an antibody to an HNA-3a or HNA-3b antigen within abiological sample may be carried out using Western blot analysis withimmunoblotting, immunocytochemistry, immunohistochemistry, dot blotanalysis, flow cytometry, ELISA assays or RIA assays. These techniquesand other approaches are conventional in the art (See Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratories(New York, 1989).

In addition, microcytotoxicity assays may be used to detect HNA-3a orHNA-3b in a biological sample. Microcytotoxicity assays involve themixing of pure neutrophils derived from the recipient or donor with wellcharacterized typing antibodies that are HNA-3a or HNA-3bimmunoreactive. The mixture is incubated for a sufficient time to allowthe antibodies to bind to the neutrophil surface HNA antigens. This isfollowed by addition of complement, which may be derived from, forexample, rabbit serum. The addition of complement results in complementfixation and any cells with antibody bound to their cell surface willlyse due to the complement fixation reaction. The quantity of lysedcells can be measured using a variety of different methods. For example,a vital dye which is excluded from live cells but stains dead cells,such as trypan blue, can be added to the sample and the number of deadcells versus live cells can be determined.

Methods of Detecting HNA-3 Nucleic Acids

The invention provides for methods of detecting HNA-3a nucleic acids ina biological sample using oligonucleotide probes that hybridize to afragment of the nucleic acid sequence of SEQ ID NO: 3. The inventionprovides for methods of detecting HNA-3b nucleic acids in a biologicalsample using oligonucleotide probes that hybridize to a fragment of thenucleic acid sequence of SEQ ID NO: 4. Hybridization of the HNA-3a orHNA-3b specific oligonucleotide probes may be detected using NorthernBlot analysis, Southern Blot analysis, slot-blot analysis or in situhybridization analysis or any other methods convention in the art, suchas those techniques described in Sambrook et al. (Molecular Cloning: ALaboratory Manual, Cold Springs Harbor Laboratories (New York, 1989).

Preferred oligonucleotide probes are those which hybridize to sequenceswithin the HNA-3a gene that encode the HNA-3a epitope. For example,preferred probes may hybridize to the nucleotides encoding the aminoacid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42. In addition,the probes of the invention include those which hybridize to introns or5′ and 3′ untranscripted regions of the gene encoding HNA-3a.

Preferred oligonucleotide probes are those which hybridize to sequenceswithin the HNA-3b gene that encode the HNA-3b epitope. For example,preferred probes may hybridize to the nucleotides encoding the aminoacid sequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26. In addition, theprobes of the invention include those which hybridize to introns or 5′and 3′ untranscripted regions of the gene encoding HNA-3b.

The oligonucleotide probes may be labeled for detection of hybridizationwith the DNA extracted from the biological sample. The probes maycomprise a radioactive label such as ³H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I. Inaddition, the labels may be a fluorescent or chemiluminescent compound,such as fluorescein isothiocyanate, phycoerythrin, rhodamine, orluciferin. The labels may be enzymes such as alkaline phosphatase,β-galactosidase, biotin and avidin or horseradish peroxidase (Bayer etal., Meth. Enz., 184:138-163 (1990)).

An array or microarray refers to a collection of DNA probes or DNAfragments attached to a solid surface, such as glass, plastic orsilicon-chip, forming an array for the purpose of expression profilingor monitoring expression level of many genes simultaneously. Arrays ofoligonucleotide probes may be used to detect the HNA-3a or HNA-3b DNA ina biological sample. Preferred arrays include probes that hybridize toHNA-1 and/or HNA-2. In addition, arrays that include probes thathybridize to HNA-1, HNA-2 and HLA are preferred. Commercial arrays maybe used to detect HNA-3a or HNA-3b that include probes that hybridize toCTL2 such as Affymetrix probe sets nos. 58800, 48798 and 56340 whichdetect SLC44A2 on array nos. U95-C and U95-B, probe set nos. 225175 and224609 which detect SLC44A2 on array no. U133-B, and probe set nos.225175 and 224609 which detect SLC44A2 on array no. U133 Plus 2. Thearrays of the invention include microarrays, DNA chips, bead arrays,gene chips and biochips.

The oligonucleotide probes may be attached to solid substrates such asmembranes, beads, filters, glass, silicon, metal, metal-alloy, anopore,polymeric, nylon or plastic. The substrates may be chemically treatedwith chemical prior to attaching probes to enhance binding or to inhibitnonspecific binding during use. Exemplary treatments include coatingglass slides with coating of aminoalkyl silanes or polymeric materialssuch as acrylamide or proteins. The probes may be covalently ornon-covalently attached to the substrate.

The invention also provides for methods of detecting HNA-3a or HNA-3b ina biological sample using an amplification method such as polymerasechain reaction and at least one oligonucleotide primer specific for afragment of the nucleic acid sequence encoding HNA-3a (SEQ ID NO: 1) orHNA-3b (SEQ ID NO: 2).

As used herein, “polymerase chain reaction” or “PCR” means a processsuch as described in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202for the amplification of a segment of DNA using at least two primers anda DNA polymerase. Other nucleic acid amplification methods stranddisplacement assay 3 (SDA, BD ProbeTec™, isothermal amplificationmethods such as helicase-dependent amplification (HDA) and isothermalreverse transcription-thermophilic helicase-dependent amplification(RT-tHDA), rolling circle amplification (RCA) and loop-mediatedisothermal amplification (LAMP). These methods may be carried out usingtechniques standard in the art. The invention also contemplates usingsequencing analysis to confirm the identity of DNA fragments amplifiedusing PCR.

In the methods of the invention, PCR may be carried out using a “PCRreaction mixture” which is a mixture suitable for carrying out PCR. ThePCR reaction mixture will contain a suitable amount of a thermostableDNA polymerase, a linear or circular template DNA, preferablydouble-stranded DNA, to be amplified, a pair of oligonucleotide primerssuch that one of the primers is configured for annealing to one strandof the template and the other primer is configured for annealing to theother or complementary strand of the template, ATP, suitable amounts ofeach of the four deoxyribonucleoside triphosphates (dNTPs), and buffers,salts such as MgCl₂, preservatives, reducing agents, and water as may berequired.

The oligonucleotide primers of the invention will be designed tospecifically amplify the nucleic acid encoding the HNA-3a epitope or theHNA-3b epitope. When designing the oligonucleotide primers, the lengthof a primer depends upon its (A+T) content, and the Tm of its partner.In addition, the primer should be complex enough to decrease thelikelihood of the primer annealing to sequences other than the chosentarget. The methods of the invention may utilize primers ranging inlength from 10-30 nucleotides, preferably the primers will be 17nucleotides in length. Generally, a 40%-60% G+C content is recommendedfor the primers, avoiding internal secondary structure and longstretches of any one base. In addition, primers should not anneal toregions of secondary structure (within the target) having a highermelting point than the primer.

Preferred oligonucleotide primers of the invention for genotyping HNA-3aphenotype include primers that amplify the nucleotides encoding aminoacid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42. In addition,the primers of the invention include those which amplify fragments ofSEQ ID NO: 3 that are within introns or the 5′ and 3′ untranscriptedregions of the gene encoding HNA-3a. Exemplary primers include senseprimer 5′ AGT GGC TGA GCT TCG 3′ (SEQ ID NO: 48) and antisense primer 5′GTG CGC CAA TAT CCT CAC TTG 3′ (SEQ ID NO: 50).

Preferred oligonucleotide primers of the invention for genotyping HNA-3bphenotype include primers that amplify the nucleotides encoding aminoacid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26. The invention alsocontemplates primers the amplify a fragment of SEQ ID NO: 4 thatcomprise codon 154 of SEQ ID NO: 4. In addition, the primers of theinvention include those which amplify fragments of SEQ ID NO: 4 that arewithin introns or the 5′ and 3′ untranscripted regions of the geneencoding HNA-3b. Exemplary primers include sense primer 5′ GAG TGG CTGTGC TTC A 3′ (SEQ ID NO: 49) and antisense primer 5′ GTG CGC CAA TAT CCTCAC TTG 3′ (SEQ ID NO: 50).

The invention also contemplates methods of detecting HNA-1 nucleic acidin a biological sample in addition to detecting HNA-3 nucleic acid.HNA-1 nucleic acids may be detected using oligonucleotide probes oroligonucleotide primers that detect the unique HNA-1 epitopes(polymorphisms) at nucleotides 141, nucleotide 147, nucleotide 227,nucleotide 277 or nucleotide 349 (HNA-1a vs. HNA-1b) and nucleotide 266(HNA-1c vs. HNA-1b). The invention further contemplates methods ofdetecting HNA-2 nucleic acid in a biological sample in addition todetecting HNA-3 nucleic acid. Since HNA-2 only has one allele, theexpression of the HNA-2 can be detected with oligonucleotide probes orprimers homologous to any coding region.

Deoxyribonucleoside triphosphates (dNTPs) include 2′-deoxyadenosine5′-triphosphate (dATP), 2′-deoxycytidine 5′-triphosphate (dCTP),2′-deoxyguanosine 5′-triphosphate (dGTP), and 2′-deoxythymidine5′-triphosphate (dTTP). Generally, the concentration of dNTP in the PCRreaction is about 200 μM. It is important to keep the four dNTPconcentrations above the estimated Km of each dNTP (10 μM-15 μM) andbalanced for best base incorporation. Lowering the concentrations ofdNTP and magnesium ion by an equal molar concentration can improvefidelity. Modified dNTPs (dig-11-dUTP, 5-bromo-dUTP, inosine,biotin-11-dUTP, biotin-16-dUTP and 7-deaza dGTP) and 2′-deoxyuridine5′-triphosphate (dUTP) also may be used.

Kits

The invention provides for kits to carry out any of the methods of theinvention. Kits according to the invention comprises components fordetecting HNA-3a or HNA-3b specific antibodies in a biological sample.The kit can comprise an isolated or recombinant HNA-3a or HNA-3bpolypeptide or an antigenic fragment thereof that forms a complex withHNA-3a or HNA-3b specific antibodies in a biological sample and a knownHNA-3a or HNA-3b specific antibody for a positive control. The inventionfurther provides for kits for detecting antibodies specific for HNA-1and HNA-2 in addition to antibodies specific for HNA-3a or HNA-3b, whichwill contain Fc-γ receptor IIIb or antigenic fragments thereof for HNA-1detection and CD177 or antigenic fragments thereof for detection ofHNA-2 and known antibodies that are specific for HNA-1 and HNA-2. Theinvention further provides for kits for detecting antibodies specificfor HNA-4 and/or HNA-5 in addition to antibodies specific for HNA-3a orHNA-3b, which will contain CD11b (CR3) or antigenic fragments thereoffor HNA-4 detection and CD11a (LFA-1) or antigenic fragments thereof fordetection of HNA-5 and known antibodies that are specific for HNA-4 andHNA-5. Furthermore, the invention provides for kits for detectingantibodies specific for HLA in a biological sample in addition toantibodies specific for HNA, which will contain polypeptides containingthe HLA antigen and known antibodies that are specific for HLA.

Kits useful for detecting antibodies specific for HNA-3a or HNA-3b andoptionally antibodies specific for other HNA and/or HLA antigens mayfurther comprise any components necessary to carry out the detectionassays that are conventional in the art. For example, the kits maycomprise the components necessary to carry out SRID, ELISA, HAI, MAIGAassay, GIIFT, MLAT, and GAT.

Kits according to the invention comprises components for detectingHNA-3a or HNA-3b in a biological sample. The kit can comprise anantibody that specifically binds to HNA-3a or HNA-3b, and an isolated orrecombinant protein or a peptide comprising the HNA-3a or HNA-3b epitopefor the antibody to use as a positive control. The invention furtherprovides for kits for detecting HNA-1 and HNA-2 in addition to HNA-3a orHNA-3b, which will contain antibodies specific for HNA-1 and/or HNA-2and recombinant proteins or peptides corresponding to the HNA-1 andHNA-2 epitopes. Furthermore, the invention provides for kits fordetecting HLA in a biological sample in addition to HNA, which willcontain antibodies specific for HLA and recombinant protein or peptidesthat correspond to the HLA epitope.

Kits useful for detecting HNA-3a or HNA-3b and optionally other HNAand/or HLA antigens may further comprise any components necessary tocarry out the detection assays that are conventional in the art. Forexample, the kits may comprise buffers, loading dyes, gels such aspolyacrylamide gels and molecular weight markers preparing SDS-PAGE gelsto carry out Western blots. The kits may also comprise filters,membranes blocking buffers, control buffers, isotype control antibodies,wash buffers or buffers and reagents for detection to carry outimmunoblotting or dot blotting analysis such as labeled secondaryantibodies. The kit may also comprise fixing reagents, blocking buffers,control buffers, wash buffers, staining dyes and detection reagentsincluding anti-idiospecific antibodies to carry out immunocytochemistryor immunohistochemistry. Furthermore, the kits may comprise thenecessary reagents and tools to carryout flow cytometry, ELISA assays,RIA assays or microtoxicity assays.

Kits according to the invention comprise components for detecting HNA-3aor HNA-3b nucleic acid in a biological sample. The kit will compriseoligonucleotide probes that hybridize to a fragment of HNA-3a nucleicacid of SEQ ID NO: 3 and a fragment of the nucleic acid of SEQ ID NO: 3that hybridizes to the oligonucleotide probes to use as a positivecontrol. Alternatively, the kit will comprise oligonucleotide probesthat hybridize to a fragment of HNA-3b nucleic acid of SEQ ID NO: 4 anda fragment of the nucleic acid of SEQ ID NO: 4 that hybridizes to theoligonucleotide probes to use as a positive control. The inventionfurther provides for kits to detect HNA-1 and HNA-2 nucleic acid, inaddition to HNA-3a or HNA-3b nucleic acid in a biological sample, whichwill contain oligonucleotide probes that are specific for HNA-1 andHNA-2, and corresponding fragments of the HNA-1 and HNA-2 nucleic acidsas positive controls. In addition, the invention provides for kits fordetecting HLA nucleic acid in addition to HNA nucleic acid in abiological sample, which will contain oligonucleotide probes specificfor HLA nucleic acid and corresponding fragments of HLA nucleic acid aspositive controls.

Alternatively, the kits for detecting HNA-3a nucleic acids will compriseoligonucleotide primers for amplifying a fragment of the HNA-3a nucleicacid of SEQ ID NO: 3, and a fragment of the HNA-3a nucleic acid that isknown to be amplified by the oligonucleotide primers to serve as apositive control. The kits for detecting HNA-3b nucleic acids willcomprise oligonucleotide primers for amplifying a fragment of the HNA-3bnucleic acid of SEQ ID NO: 4, and a fragment of the HNA-3b nucleic acidthat is known to be amplified by the oligonucleotide primers to serve asa positive control. The invention further provides for kits thatcomprise oligonucleotide primers specific for HNA-1 and HNA-2 nucleicacids, and the fragments of the nucleic acids of HNA-1 and HNA-2 thatare known to be amplified by the oligonucleotide primers. In addition,the invention provides for kits containing oligonucleotide primersspecific for HLA nucleic acid in addition to oligonucleotide primersspecific for HNA nucleic acids and a fragment of the HLA nucleic acidthat is amplified by the oligonucleotide primers.

The kits of the invention may also comprise the components necessary tocarry out PCR or other amplification methods. For example, the kit maycontain one or more of the following: Taq polymerase or anotherthermostable polymerase, ATP, suitable amounts of each of the fourdeoxyribonucleoside triphosphates (dNTPs), and buffers, salts such asMgCl2, preservatives, reducing agents or water.

Kits useful for detecting HNA-3a or HNA-3b nucleic acids and optionallyother HNA and/or HLA nucleic acids may further comprise any componentsnecessary to carry out the detection assays that are conventional in theart. For example, the kits may comprise the reagents necessary forextracting the nucleic acids from the biological sample. The kits maycomprise buffers, loading dyes, gels, molecular weight markers,membranes, filters, blocking buffers and detection reagents for NorthernBlot analysis, Southern Blot analysis, slot-blot analysis or in situhybridization analysis and any other methods convention in the art, suchas those techniques.

The present invention is illustrated by the following examples that arenot intended to limit the invention. Example 1 describes isolation ofgranulocytes from donor blood. Example 2 described the method used toobtain HNA-3a positive and negative plasma. Example 3 describesbiotinylation of granulocyte surface proteins. Example 4 describes amethod of incubating granulocytes with plasma. Example 5 describesfluorescence activated cell sorting using flow cytometry. Example 6describes a method of carrying out immunoprecipitation using magneticbeads. Example 7 describes a method of carrying out SDS-PAGE and Westernblotting. Example 8 describes a method of using Fourier transform ioncyclotron resonance mass spectrometry (FTICR-MS). The experimentsdescribed in Examples 1-8 were used to identify the amino acid sequencesof HNA-3a and HNA-3b, and these methods may be used to carry out themethods of the invention. Example 9 describes the heterologousexpression of HNA-3a and HNA-3b, and these polypeptides may be used inthe methods of the invention. Example 10 describes heterologousexpression of HNA-3a peptide fragments, and these peptide fragments wereused to map the epitope of HNA-3a, and these peptides may be used in themethods of the invention. Example 11 describes affinity purification ofHNA-3a antibodies from human blood plasma. Example 12 describedidentification of antigenic fragments of HNA-3a, and these fragments maybe used to carry out the methods of the invention. Example 13 describesa method of genotyping for HNA-3a and HNA-3b, which may be used to carryout the methods of the invention. Lastly, Example 14 describes methodsof making anti-HNA-3 antibodies, and these antibodies may be used tocarry out the methods of the invention.

EXAMPLES Example 1 Granulocyte Isolation

Selected donor blood was mixed with 1.25% EDTA and 0.5% dextran. Aftersedimentation, the supernatant was further used for Ficoll densitycentrifugation. After washing of the obtained pellet, a haemolysis ofthe erythrocytes took place. Remaining granulocytes were washed andserved in various cell concentrations as starting material.

Example 2 Obtaining HNA-3a Positive and Negative Plasma

Selected donor blood was mixed with 1.25% EDTA, then the cells wereseparated by centrifugation. The supernatant served as correspondingplasma.

Example 3 Biotinylation of Granulocyte Surface Proteins

For the analysis by Western blots, the purified granulocytes werebiotinylated using the EZ-Link Sulfo-NHS-LC-LC-biotin (PIERCE; Rockford,Ill.).

Example 4 Incubation of the Granulocytes with Plasma

Granulocytes (biotinylated or non-biotinylated) were incubated withHNA-3a-positive or -negative plasma for at least 30 minutes at 37° C.After washing the batch, either a FACS analysis of whole cells tookplace, or the cell lysis by means of buffer containing Triton-X100 wascarried out. After centrifuging off the cell debris, the supernatantcontaining protein was analyzed.

Example 5 Fluorescence Activated Cell Sorting (FACS Analysis) FlowCytometry

Isolated granulocytes of HNA-3a-positive and -negative donors wereincubated with plasma (with and without anti-HNA-3a antibodies) andwashed. Subsequently, the cells were incubated with fluoresceinisothiocyanate (FITC) labeled rabbit F(ab′)2-anti-human IgG. Afterwashing, the cell suspension was examined with FACS to determine for thefluorescence intensity of the granulocytes. High intensities pointed toHNA-3a-positive results.

Example 6 Immunoprecipitation by Means of Magnetic Beads

Protein-G coated magnetic beads were coupled with anti-human IgG (Fcspecific). After washing, the beads were incubated with the supernatantcontaining protein, as described in Example 4. After renewed washing anddemagnetizing of the beads, the proteins were eluted using sample buffercontaining SDS, (for analysis via SDS-PAGE and Western blot) or digesteddirectly in buffer containing trypsin (for analysis via Fouriertransform ion cyclotron resonance mass spectrometry (FTICR-MS)).

Example 7 SDS-PAGE/Western Blot

The eluent of the immunoprecipitation (biotinylated batches) wasseparated by SDS-PAGE (7.5% separation gels) and blotted onnitrocellulose membrane. For coupling to the biotinylated proteins, themembrane, after blocking and washing steps, was incubated with alkalinephosphatase (AP) bonded to streptavidin. The detection took place by theaddition of NBT (Nitro-Blue Tetrazolium Chloride)/BCIP(5-Bromo-4-Chloro-3′-Indolyphosphate p-Toluidine Salt) for 1-15 minutes.

Example 8

Fourier Transform Ion Cyclotron Resonance (FTICR-MS)

The tryptic digestion of the immunoprecipitated proteins (notbiotinylated batches) was pre-cleaned using C18 material (ZipTip) andanalyzed by MS. The evaluation of the peptide spectra took place by databank comparisons using of SEQUEST Sorcerer- and Scaffold2 software (Databank: uniprot-sprot-human_re 154).

Example 9 Heterologous Expression of HNA-3a and HNA-3b

A cDNA clone having a DNA sequence encoding the HNA-3a protein (SEQ IDNO: 1) was expressed in E. coli and in CHO cells. The synthesizedprotein was separated with SDS-PAGE gel electrophoresis (as described inExample 7) and the specificity was shown by binding of human anti-HNA-3aantibodies in Western blot. An analogous procedure was carried out for acDNA clone with the DNA sequence for the expression of the proteinHNA-3b (SEQ ID NO: 2).

The HNA-3a protein was expressed with a His-tag to allow forpurification. In addition, solid phase ELISA was used to demonstratebinding of the recombinant human anti-HNA-3a or HNA-3b protein with itsrespective antibody.

Example 10 Heterologous Expression of Peptide Fragments

HNA-3a DNA fragments were cloned using the pGEX-2TK vector (GEHealthcare, Chalfont St Giles, UK) and the restriction enzymes BamHI andHindIII (Roche, Basel, Switzerland). The cDNA clone NM_(—)020428.2(OriGene, Rockville, Md.) was used as template DNA to generateGlutathione S-Transferase gene region (GST) fusions. DNA fragmentsencoding amino acids 22-231 of SEQ ID NO: 1 (denoted as“HNA-3a(22-231)”), and amino acids 145-167 of SEQ ID NO: 1 (denoted as“HNA-3a(145-167)”) were inserted into the vector JHC27 to encodeGST-HNA-3a fusion peptides. DNA fragments encoding amino acids 114-164of SEQ ID NO: 1 (denoted as “HNA-3a(114-164)”) were inserted into thepTB25 vector to encode the GST fusion peptide GST-HNA-3a(114-164).

E. coli BL21-Gold (DE3) cells (Stratagene, La Jolla, Calif.) weretransformed with the above described vectors and grown at 37 C in YTGmedium containing 100 μg/ml ampicillin to an OD600=0.7. Subsequently,the cells were incubated with Isopropyl β-D-1-thiogalactopyranoside(IPTG) (1 mM; 1 h), followed by centrifugation (7,000 g; 10 min) andwashing in ice-cold PBS. The cells were then sonicated in denaturingbuffer (8 M Urea), centrifuged (12,000 g) and the supernatant wasdialyzed against denaturing-buffer (Tris-glycerin). The fusion proteinsupernatants were loaded onto a glutathione sepharose column and washedwith PBS. A solution of 50 mM Tris-HCl (pH 8) containing 10 mM reducedglutathione was used for elution of the GST-HNA-3a fusion peptides.

Example 11 Affinity Purification of HNA-3a Antibodies from Human BloodPlasma

For affinity purification of HNA-3a antibodies from human blood plasma,GST-HNA-3a(114-164) was produced as described in Example 10),concentrated and dialyzed against coupling buffer (0.2 M NaHCO₃, 0.5 MNaCl, pH 8.3). A HiTrap NHS-activated HP column (GE Healthcare, Uppsala,Sweden) was equilibrated with 6 ml ice-cold 1 mM HCl; and subsequently,1 ml of the concentrated GST-HNA-3a(114-164) peptide was injected (3mg/ml) onto the column. After 30 minutes incubation at 25° C., thecolumn was washed according to manufacturer's instructions.

Human plasma known to be positive for HNA-3a antibodies was diluted inwash buffer (1:25) (20 mM NaH2PO4, 150 mM NaCl, pH 7.4) and 50 ml wasloaded onto the column at a flow rate of 0.8 ml/min. After washing withwashing buffer, the antibodies were eluted from the column using 10 ml0.1 M glycine buffer (pH 2.7). Aliquots of the collected fractions (750μl) were mixed with 250 μl neutralization buffer (1 M Tris-HCl, pH 9).Fractions containing a protein concentration greater than 50 μg/ml werepooled and dialyzed against wash buffer.

A Granulocyte Activation Assay was used to test whether HNA-3a inducedgranulocyte aggregation. Granulocytes isolated from HNA-3a positivedonors (as described in Example 1) were incubated with human plasma (30min, 37° C.) known to contain anti-HNA-3a antibodies, and subsequentlywashed (140 g, 5 min). Bound antibodies were obtained using system foracid elution according to the manufacturer's instructions (BAG, Lich,Germany).

The HNA-3a specific antibodies, of the eluted antibodies obtained byaffinity purification in combination with the GST-HNA-3a(114-164)peptide in the Granulocyte Activation Assay. The negative controls forthis assay were the GST-fusion protein alone, a control serum containingHNA-3b antibodies and a negative control antibody (no HNA-3 antibodies).HNA-3a antibodies activated granulocytes to aggregation only in itsnative form and had little effect in denatured formed. Fixed granulocyteagglutinates were not positive.

Example 12 Identification of Antigenic Fragments of HNA-3a

In order to map the epitope of the HNA-3a amino acids sequence (SEQ IDNO: 1), recombinant peptides comprising extracellular fragments of theHNA-3a amino acid sequence were generated as GST-fusion peptides asdescribed in Example 10. Reactivity of these peptides with HNA-3a seraas determined by Western blot is provided in Table 1 below

Amino acids of HNA-3a Reactivity SEQ ID NO: (SEQ ID NO: 1) with HNA-3a27  55-231 ++ 14  55-183 ++ 28  55-105 −− 29 105-153 −− 30 155-200 −− 31190-231 −− 32 150-159 −− 33 144-167 (+) 34 134-174 ++ 35 124-183 ++ 36114-194 ++ 37 105-200 ++ 38  94-214 ++ 39  84-220 ++ 40  44-164 ++ 41 75-164 ++ 42  94-164 ++ 16 114-164 ++ 43 134-164 (+) 44 142-183 −− 45142-200 −− 46 142-222 −− 47 142-231 −−

Isolated granulocytes from a representative HNA-3a positive (HNA-3a+)donor and a representative HNA-3a negative donor (HNA-3a−) wereincubated with plasma known to contain HNA-3a antibodies (+) and withplasma known to be free of HNA-3a antibody (−). HNA-3a protein was thenimmunoprecipitated using anti-human IgG, coupled with Protein G coupledmagnetic beads. For each donor/plasma combination was a sample wasdeglycosylated with Peptide-N-Glycosidase F (PNGase F).

The immunoprecipitated proteins were separated by SDS-PAGE andtransferred to nitrocellulose using procedures well known in the art,and analyzed by immunoblotting with a HNA-3a+ and HNA-3a− plasma. Theproteins were initially visualized using alkaline phosphatase conjugatedto streptavidin (a) or anti-human IgG (b) and then incubated withNBT/BCIP for detection.

The GST-fusion peptides comprising amino acids 145-167 of SEQ ID NO: 1(HNA-3a(145-167); SEQ ID NO: 48) and amino acids 55-231 of SEQ ID NO: 1(HNA-3a(55-231); SEQ ID NO: 27) were analyzed using an immunoblot. A 50kDa band represents the GST-HNA-3a(55-231) peptide bound to an antibodypresent in HNA-3a positive sera. A smaller band of 36 kDa represents theGST-HNA-3a(145-167) peptide bound to an antibody present in HNA-3apositive sera. Only antibodies from HNA-3+ plasma reacted with theHNA-3a fusion peptides. Antibodies from the HNA-3a− plasma did not reactwith the HNA-3a fusion proteins. The binding to the longer HNA-3a aminoacids 55-231 peptide is shown for two HNA-3a positive plasmas and thebinding for the smaller fragment HNA-3a amino acids 145-167 is displayedfor HNA-3a positive plasma.

The GST-fusion peptide HNA-3a amino acids 145-167 also inducedaggregation in HNA-3a positive granulocytes.

This analysis demonstrated that the critical minimum antigenic fragmentof the HNA-3a polypeptide sequence is amino acids 154-164 of SEQ ID NO:1 (SEQ ID NO: 25).

This method may be used to identify the antigenic fragments of HNA-3a orany other antigen such as HNA-1, HNA-2, HNA-5 or HLA.

Example 13 Genotyping for HNA-3a and HNA-3b

PCR for HNA-3 polymorphisms were carried out as follows. Aliquots of50-100 ng DNA were amplified using 0.5 pmol allele-specific senseprimers (5′-AGT GGC TGA GGT GCT TCG-3; SEQ ID NO: 49; HNA-3a) or 5′-GAGTGG CTG AGG TGC TTC A-3′; SEQ ID NO: 50; HNA-3b)) and a partial intronicantisense primer (5′-GTG CGC CAA TAT CCT CAC TTG-3′(SEQ ID NO: 51)).Polymerase chain reaction (PCR) was performed with 0.2 mmoldeoxyribonucleotide triphosphate and 2.0 units Hot Start Taq DNAPolymerase (GeneCraft, Germany) on a Thermal Cycler (GeneAmp PCR System2700, Applied Biosystems, Germany) in a total volume of 20 μL. Afterheating at 95° C. for 10 minutes, 2-step PCR was performed under thefollowing conditions: denaturing (30 seconds, 95° C.), annealing (40seconds, 64° C.), extension (30 seconds, 72° C.) for 10 cycles,denaturing (30 seconds, 95° C.), annealing (30 seconds, 61° C.),extension (30 seconds, 72° C.) for 20 cycles, and final extension (5minutes, 72° C.). As internal positive control, 0.0625 pmol human growthhormone (hGH) primers amplifying a 439-bp fragment of the hGH gene wereused (5′-CAG TGC CTT CCC AAC CAT TCC CTT A-3′ (SEQ ID NO: 52), 5′-ATCCAC TCA CGG ATT TCT GTT GTG TTT C-3′ (SEQ ID NO: 53)). PCR products (291bp) were analysed on 1.5% agarose gels using Tris borate EDTA buffer(TBE-buffer; 5 Prime, Germany).

This method may be used to genotype for any antigen allele such asHNA-1, HNA-2, HNA-4, HNA-5 or HLA.

Example 14 Methods of Making anti-HNA-3 Antibodies

Antibodies specific for HNA-3a or HNA-3b protein may be obtained byimmunization with peptide comprising a particular HNA-3 epitope.Suitable procedures for generating antibodies include those described inHudson and Hay, Practical Immunology, 2nd Edition, Blackwell ScientificPublications (1980). Exemplary antigenic fragments that may be used togenerate HNA-3a specific antibodies include SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41and SEQ ID NO: 42. Exemplary antigenic fragments that may be used togenerate HNA-3b specific antibodies include SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQID NO: 26.

In one procedure for the production of antibodies, animals (typicallymice or rabbits) are injected with a HNA-3a or HNA-3b epitope containingpeptide and the resulting polyclonal antibodies in the serum areisolated. In addition, those animals with sufficient serum titer levelsas determined by ELISA are selected for hybridoma production. Spleens ofimmunized animals are collected and prepared as single cell suspensionsfrom which splenocytes are recovered. The splenocytes are fused to mousemyeloma cells (such as Sp2/0-Ag14 cells; ATCC no. CRL-1581), allowed toincubate in DMEM with 200 U/ml penicillin, 200 g/ml streptomycinsulfate, and 4 mM glutamine, and then incubated in HAT selection medium(Hypoxanthine; Aminopterin; Thymidine). After selection, the tissueculture supernatants are taken from each well containing a hybridoma andtested for anti-HNA-3a or HNA-3b antibody production by ELISA.

Alternative procedures for obtaining anti-HNA-3a or anti-HNA-3bantibodies may also be employed, such as the immunization of transgenicmice harboring human Ig loci for the production of human antibodies, andthe screening of synthetic antibody libraries, such as those generatedby mutagenesis of an antibody variable domain.

Furthermore, human antibodies can be produced from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks etal., J. Mol. Biol. 222:581 (1991). These processes mimic immuneselection through the display of antibody repertoires on the surface offilamentous bacteriophage, and subsequent selection of phage by theirbinding to an antigen of choice. One such technique is described in PCTApplication no. PCT/US98/17364, which describes the isolation of highaffinity and functional agonistic antibodies for MPL- and msk-receptorsusing such an approach.

These methods are used to generate antibodies specific for any antigenicprotein such as HNA-1, HNA-2, HNA-4, HNA-5 or HLA.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe presently preferred embodiments thereof. Consequently, the onlylimitations which should be placed upon the scope of the invention arethose which appear in the appended claims.

What is claimed is:
 1. A method of determining whether a donor tissueintended for transplant or transfusion is likely to be rejected in ahuman recipient wherein the human recipient expresses antibodies thatspecifically bind the amino acid sequence of SEQ ID NO: 1 or anantigenic fragment thereof, comprising a) obtaining a sample of thetissue intended for transplant or transfusion, b) extracting nucleicacids from the sample, c) contacting the nucleic acids witholigonucleotide probes that hybridize to a fragment of SEQ ID NO: 3, andd) detecting hybridization of the probe to the nucleic acids, whereinhybridization of the probe indicates the presence of an HNA-3a nucleicacid wherein HNA-3a nucleic acid comprises the nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 1 or an antigenicfragment thereof, and wherein the presence of HNA-3a nucleic acid in thesample indicates that the sample is likely to be rejected in a humanrecipient that expresses HNA-3a specific antibodies.
 2. The method ofclaim 1 wherein the oligonucleotide probe comprises a nucleotidesequence encoding an amino acid sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:42.
 3. The method of claim 1 further comprising one or more of the stepsof d) contacting the sample with an oligonucleotide probe thathybridizes to a fragment of the HNA-1 nucleic acid, wherein the HNA-1nucleic acid comprises the nucleotide sequence of SEQ ID NO: 5, e)contacting the sample with an oligonucleotide probe that hybridizes to afragment of the HNA-2 nucleic acid, wherein the HNA-2 nucleic acidcomprises the nucleotide sequence of SEQ ID NO: 7, f) contacting thesample with an oligonucleotide probe that hybridizes to a fragment ofthe HNA-4 nucleic acid, wherein the HNA-4 nucleic acid comprises thenucleotide sequence of SEQ ID NO: 9, g) contacting the sample with anoligonucleotide probe that hybridizes to a fragment of the HNA-5 nucleicacid, wherein the HNA-5 nucleic acid comprises the nucleotide sequenceof SEQ ID NO: 11, or h) contacting the sample with an oligonucleotideprobe that hybridizes to a fragment of a nucleotide sequence encoding anHLA antigen, and i) detecting the hybridization of the probe to thenucleic acids, wherein hybridization of any of the probes indicates thepresence of any one of HNA-1, HNA-2, HNA-3b, HNA-4, HNA-5 or HLA nucleicacid in the sample and the presence of any one of HNA-1, HNA-2, HNA-4,HNA-5 or HLA nucleic acid in the sample indicates that the sample islikely to be rejected in a human recipient.
 4. The method of claim 3wherein the fragment of the HNA-1 nucleic acid comprises at least anyone of nucleotides 141, nucleotide 147, nucleotide 226, nucleotide 227,nucleotide 277 or nucleotide 349 of SEQ ID NO:
 5. 5. A method ofdetermining whether a donor tissue intended for transplant ortransfusion is likely to be rejected in a human recipient wherein thehuman recipient has antibodies that specifically bind to the amino acidsequence of SEQ ID NO: 1 or an antigenic fragment thereof, comprising a)obtaining a sample from the tissue, b) extracting nucleic acids from thesample, c) amplifying a fragment of HNA-3a nucleic acid of SEQ ID NO: 3from the extracted nucleic acids using at least one oligonucleotideprimer specific for HNA-3a nucleic acid, and c) detecting the fragmentof HNA-3a nucleic acid in the sample, wherein the presence of HNA-3anucleic acid in the sample indicates that the sample is likely to berejected in a human recipient that has HNA-3a specific antibodies. 6.The method of claim 5 wherein the fragment of HNA-3a nucleic acidencodes an amino acid sequence selected from the group consisting of SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:
 42. 7. The method ofclaim 5 further comprising one or more of the steps of d) amplifying afragment of HNA-1 nucleic acid wherein the HNA-1 nucleic acid comprisesthe nucleotide sequence of (SEQ ID NO: 5 using at least oneoligonucleotide primer specific for HNA-1 nucleic acid, e) amplifying afragment of HNA-2 nucleic acid wherein the HNA-2 nucleic acid comprisesthe nucleotide sequence of SEQ ID NO: 7 using at least oneoligonucleotide primer specific for HNA-2 nucleic acid, f) amplifying afragment of HNA-4 nucleic acid wherein the HNA-4 nucleic acid comprisesthe nucleotide sequence of SEQ ID NO: 9 using at least oneoligonucleotide primer specific for HNA-4 nucleic acid, g) amplifying afragment of HNA-5 nucleic acid wherein the HNA-5 nucleic acid comprisesthe nucleotide sequence of SEQ ID NO: 11 using at least oneoligonucleotide primers specific for HNA-5 nucleic acid, or h)amplifying a fragment of HLA nucleic acid using at least oneoligonucleotide primer specific for HLA nucleic acid, and h) detectingthe fragment of any HNA-1, HNA-2, HNA-4, HNA-5 or HLA nucleic acid inthe sample, wherein the presence of any one of HNA-1, HNA-2, HNA-4,HNA-5 or HLA nucleic acid in the sample indicates that the sample islikely to be rejected in a human recipient.
 8. The method of claim 5wherein the fragment of the HNA-1 nucleic acid-comprises at least anyone of nucleotides 141, nucleotide 147, nucleotide 226, nucleotide 227,nucleotide 277 or nucleotide 349 of SEQ ID NO: 5.